Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Soluble TREM2 inhibits secondary nucleation of Aβ fibrillization and enhances cellular uptake of fibrillar Aβ

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer’s disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-β (Aβ) into plaques, particularly Aβ40 and Aβ42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aβ fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aβ, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aβ40 and Aβ42, even at a high micromolar concentration, while it does bind to fibrillar Aβ42 and Aβ40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aβ, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2–Aβ fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake.

Alzheimer’s disease (AD) is the most common form of dementia and features the neuropathological hallmarks of extracellular Aβ plaques and intraneuronal tau neurofibrillary tangles (1, 2). Human genetic studies on heritable mutations in APP and PSEN causing early-onset familial AD (3) argue that pathogenic Aβ drives tau neurofibrillary tangle formation; in contrast, mutations in MAPT do not lead to Aβ pathology nor cause AD, but rather a rare genetic form of early-onset primary tauopathy (4). In support of the molecular genetics, a recent cross-sectional study in postmortem human AD brain samples demonstrated the presence and correlation of robust prion bioactivity for Aβ and tau proteins in nearly all cases (5), suggesting that even at death, Aβ in prion conformations are active in the late stages of disease. Together, these data establish the importance of pathogenic Aβ throughout AD progression and highlight the urgent need to better understand the cellular and molecular mechanisms that mitigate Aβ’s role in pathogenesis.Microglia are the innate immune effector cell in the brain with myriad functions in healthy aging and neurological diseases. Recent human genetic studies have discovered mutations in several genes encoding microglia-specific proteins that increase risk for AD, thus supporting the notion that microglia are central to AD pathogenesis. Genetic variants of triggering receptor expressed on myeloid cells 2 (TREM2), a cell-surface receptor expressed on myeloid cells and microglia, increase the risk of AD by threefold, implicating microglia and the innate immune system as important determinants in AD pathogenesis (6). TREM2 consists of an extracellular Ig-like domain, a transmembrane domain, and a cytoplasmic tail. Proteolytic cleavage of TREM2 at His157 releases soluble TREM2 (sTREM2) that can be detected in the cerebrospinal fluid (7). While the function of sTREM2 is uncertain, it is believed to promote microglia survival, proliferation, and phagocytosis, making it important for cell viability and innate immune functions in the brain (6, 8, 9). Full-length membrane-bound TREM2 binds to its adaptor protein, DAP12, on the surface of microglia to transmit downstream signaling in response to clustering induced by multivalent ligands (10). Most of the studied mutations are in the Ig-like domain of TREM2. Misfolding, retention, and aberrant shedding are postulated to be caused by some mutations, while other variants have altered ability to interact with their binding partners (8, 11, 12).The R47H mutation in TREM2 constitutes one of the strongest single allele genetic risk factors for AD. The R62H, D87N, and T96K mutations in TREM2 were also linked to AD after extensive analyses of TREM2 polymorphisms (13–16). Several in vivo studies show that TREM2 regulates polarization of microglial processes toward Aβ deposits, leading to plaque compaction and pacification in human AD brain samples and mouse models (17–19). Genetic deletion of TREM2 expression in transgenic mice injected with exogenous Aβ fibrils leads to accelerated amyloid plaque seeding (20). The prominent phenotype in plaque-associated microglia suggests that the effects of AD-risk mutations or genetic deletions are driven by loss of full-length TREM2 signaling. However, a recent in vivo study using exogenously injected recombinant sTREM2 showed reduced amyloid burden and behavioral rescue in mice (21). New clues for the potential importance of sTREM2 in AD have been revealed in clinical studies on living AD patients. sTREM2 can be measured in the cerebrospinal fluid (CSF) and it increases during early stages of AD symptomology (22, 23), suggesting that sTREM2 may be a biomarker for microglia activation. Recent studies indicate that AD patients with relatively high levels of sTREM2 in the CSF have slower rates of amyloid accumulation and reduced cognitive decline (24, 25). These human data support the hypothesis that microglia and sTREM2 play a protective role in early stages of AD progression.While most risk variants of TREM2 exist in the ligand-binding Ig-like domain, the AD-associated point mutation H157Y falls within the stalk region and is known to increase the shedding of full-length TREM2, which possibly results in higher titers of sTREM2 (6). Elevated ectodomain shedding reduces cell-surface full-length TREM2 available for TREM2-mediated phagocytosis and plaque compaction as well as down-stream signal transduction. Although more work is needed, such data begin to suggest there is a delicate balance between the functions of membrane-bound and secreted TREM2, and hence opposing cellular effects of TREM2 variants can emerge (i.e., reduced versus enhanced shedding, which result in similar phenotypic outcomes by reducing cell-surface TREM2) (6, 26).sTREM2 binds to diverse ligands, including phospholipids, apolipoproteins, DNA, and Aβ. Although the full physiological and pathological roles of these interactions remain to be revealed (11, 12, 27, 28), there is general agreement that the extracellular domain of TREM2 (sTREM2) binds to oligomeric forms of Aβ42. However, the observed apparent affinities vary over many orders-of-magnitude (7, 29–31). Most studies were conducted with dimeric Fc fusion proteins, tetrameric constructs, or biotinylated protein bound to the tetrameric streptavidin, which might artificially increase the avidity of the protein for oligomeric forms of Aβ peptides (7, 29–31). Moreover, the studies that report the highest affinities relied on biolayer interferometry or surface plasmon resonance, in which oligomeric protein constructs were immobilized on a surface and Aβ peptides were allowed to diffuse over the surface. Aβ oligomers were found to bind, but they either did not dissociate at all, or they dissociated slowly, leading to affinity estimates in the picomolar to nanomolar range (7, 30, 31). However, the extent of binding of Aβ to the surface did not saturate at concentrations that were orders-of-magnitude greater than the reported dissociation constants, suggesting that the slow off-rate was instead due to precipitation of insoluble Aβ on the bilayer surface (7). In another study, Aβ was fused to the dimeric protein glutathione S-transferase (29). Furthermore, there is inconsistency in the studies involving monomeric Aβ42, with some studies finding nanomolar to low micromolar dissociation constants for the interaction of monomeric Aβ42 and TREM2 ectodomain (29, 30), in contrast to two other studies that reported weak or no interaction (7, 31).To help elucidate the role of sTREM2 and its interaction with Aβ, we evaluated the binding of sTREM2, without any nonnative oligomerization domains added to the studied construct, to specific forms of Aβ40 and Aβ42. We used NMR to show that sTREM2 does not bind to monomeric Aβ, even at high micromolar concentrations. Next, we examined the binding of sTREM2 to fibrils, formed under well-defined conditions to provide a relatively homogenous structure, as assessed by solid-state NMR (32). Additionally, because oligomeric forms of Aβ are heterogeneous and kinetically labile, we opted to determine how sTREM2 affects the formation of intermediates in the fibrillization of Aβ and show that it has a profound effect on the secondary nucleation step of the process. We find that the R47H variant binds to Aβ40 and Aβ42 fibrils with a similar affinity and inhibits their fibrilization just as the WT sTREM2 does. Finally, we show that WT sTREM2, but not the mutant R47H, strongly enhances the uptake of Aβ fibrils in human neural and microglial cells.A second goal of this report was to define the structural underpinnings of the interaction between sTREM2 and Aβ fibrils. Although individual structures of sTREM2 and Aβ40 fibrils have been reported (8, 33), the structures of the complex are not available. The molecular surface of sTREM2 is particularly interesting with regards to its function (8, 29). The crystal structure of the ectodomain of TREM2 (TREM2_ECD) revealed an immunoglobulin fold motif with a highly asymmetric distribution of charged and hydrophobic residues. The surface of the hydrophobic and aromatic protrusion at the top of the structure (Fig. 1, red dotted area) has a highly positive electrostatic potential adjacent to it is a relatively flat surface of positively charged residues (Fig. 1, black dotted area, surface 1). Surface 1 appears suited for binding to acidic moieties (like in Protein Data Bank [PDB] ID code 6B8O) (8). R47 lies near the basic patch, consistent with the R47H mutation disrupting the conformation of the CD loop (8), which comprises a large portion of surface 1. Molecular dynamics simulations suggest that disease-promoting mutations disrupt the apolar character and electrostatic surface of this region of the protein (34). The R47H mutation is also known to disrupt sTREM2’s ability to bind to and signal in response to acidic phospholipids (29). Thus, the data indicate that this surface is important for binding or signaling in response to anionic lipids. In contrast, the determinants of binding to Aβ peptides are uncertain, with different studies coming to differing conclusions concerning the effect of AD mutants on binding or uptake of Aβ fibrils (7, 29–31). Recently, it was suggested that different surfaces might be involved in binding different TREM2 ligands (29). Indeed, sTREM2 has a second unusual, variegated electrostatic surface (surface 2 in Fig. 1), with an extended band of positively charged residues flanked by acidic patches near the top and bottom of the structure, which might interact with different binding partners. Here, we use integrative structural modeling guided by chemical cross-linking mass spectrometry (XL-MS) to map the structure of the fibrillar Aβ–sTREM2 complex, and how it is affected by the R47H substitution. The resulting model suggests that the patch of hydrophobic and basic residues on sTREM2 that contains R47 does not directly interact with Aβ40 fibrils. Instead, sTREM2 is predicted to interact with Aβ primarily via surface 2, while projecting surface 1 away from the amyloid fibrils, with implications for both cellular uptake and signaling.Open in a separate windowFig. 1.Crystal structure of sTREM2 (PDB ID code 5UD7) (8), showing electrostatic potential map of the ectodomain. The white, red, and blue colors in the map correspond to the neutral, acidic, and basic residues, respectively. The map was generated using CHIMERA v1.14 (69). The hydrophobic and aromatic protrusion in sTREM2 is highlighted with a red dashed curve (hydrophobic tip). The flat surface of basic residues adjacent to the hydrophobic tip is shown with black dashed curve (surface 1). Another patch of basic residues, opposite to surface 1, is highlighted with a yellow dashed curve (surface 2). Key residues in these three regions are indicated.     

Interferon-β regulates proresolving lipids to promote the resolution of acute airway inflammation

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-β (IFN-β) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A₄ (15-epi-LXA₄) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli–evoked lung injury, and suppresses production of IFN-β and the proresolving lipid mediators 15-epi-LXA₄ and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-β delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-β accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA₄ and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-β–mediated resolution. These findings point to a pivotal role of IFN-β in orchestrating timely resolution of neutrophil and TLR9 activation–driven airway inflammation and uncover an IFN-β–initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Acute respiratory distress syndrome (ARDS) is a common syndrome associated with high mortality in patients admitted to intensive care units (1). ARDS is characterized by diffuse alveolar damage that develops in patients with known risk factors, most commonly pneumonia, sepsis, or trauma (2, 3). The initial alveolar damage leads to recruitment of neutrophils and monocytes, which further aggravate injury (3). Treatment of the underlying cause and lung-protective ventilation are the main elements of supportive therapy (4, 5). Importantly, no therapies are available to resolve the aberrant immune responses underlying ARDS.Type I interferons, IFN-α and IFN-β, are well established to confer antiviral activities to host cells and could also regulate the inflammatory response. A delayed type I interferon response triggers the generation of proinflammatory cytokines and facilitates the recruitment of monocytes to the lung, resulting in lethal pneumonia in mice infected with SARS-CoV-1 (6) or SARS-CoV-2 (7). Type I interferons break TNF-induced tolerance to Toll-like receptor (TLR) signals on monocytes/macrophages, rendering them hyperresponsive to additional TLR signals concurrent with inflammatory activation (8). For instance, bacterial DNA (CpG DNA) or mitochondrial DNA through TLR9 impairs neutrophil phagocytosis, delays neutrophil apoptosis, and perpetuates inflammation (9, 10). In contrast, IFN-β protects against lethal polymicrobial sepsis through inhibiting IL-1 production and/or induction of IL-10 (11–13). IFN-β produced by macrophages during resolution of bacterial pneumonia facilitates removal of neutrophils from inflamed tissues and reprograms macrophages to a proresolving phenotype, thereby driving inflammatory resolution in mice (14). However, the underlying mechanisms are incompletely understood; albeit these would be essential for implementing precision treatment with IFN-β.Resolution of inflammation is an active process governed by specialized proresolving lipid and protein mediators (SPMs) (15–19). These mediators converge on select receptors, including the pleiotropic lipoxin A₄ receptor/formyl peptide receptor 2 (ALX/FPR2) (20). ALX/FPR2 plays critical roles in host defense and orchestrating inflammatory resolution (20–23). ALX/FPR2 binds multiple lipid ligands, including aspirin-triggered 15-epi-lipoxin A₄ (15-epi-LXA₄) and 17-epi-resolvin D1 (17-epi-RvD1), generated within the inflammatory microenvironment (17, 18). SPMs inhibit neutrophil recruitment, promote neutrophil apoptosis and efferocytosis, and facilitate tissue repair and return to homeostasis (17, 18, 24). Activation of ALX/FPR2 with 15-epi-LXA₄ or 17-epi-RvD1 counters TLR9-generated cues, restores impaired neutrophil function, and enhances timely resolution of airway bacterial infections (9). Since resolution of inflammation is skewed toward a proresolving lipid profile (18, 25, 26), we investigated whether IFN-β can modulate ALX/FPR2-based resolution mechanisms. Here, we report that IFN-β exerts partially overlapping, but also cooperative actions with 17-epi-RvD1 to counter TLR9-generated signals to regulate neutrophil phagocytosis and apoptosis in vitro. In mice, IFN-β facilitates clearance of bacteria, neutrophil apoptosis and efferocytosis, and promotes the resolution of acute airway inflammation, in part, by stimulating generation of proresolving lipids and activation of ALX/FPR2-centered proresolving circuits. Our results uncover a hitherto unrecognized effector mechanism by which IFN-β may facilitate resolution of ARDS.     

Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors

β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.

β-Lactamases are a major mechanism of resistance to the clinically vital β-lactam antibiotics, with >2,000 different β-lactamases reported (1). β-Lactamases are grouped into classes A, C, and D, which employ a nucleophilic serine in catalysis (serine β-lactamases, SBLs), and class B, which employ metal ions in catalysis (2). Presently, SBLs are the most important β-lactamases from a clinical perspective. SBL inhibitors (SBLi) have been developed for use in combination with a β-lactam antibiotic, with tazobactam (3), sulbactam (4), and clavulanic acid (5) being the most widely used SBLi. These SBLi all contain a β-lactam ring which reacts with SBLs to produce an acyl–enzyme complex (AEC) intermediate, as is also the case for efficient SBL substrates (Fig. 1A). With efficient substrates the β-lactam–derived AEC is readily hydrolyzed. With SBLi the reaction bifurcates at the AEC stage; in addition to hydrolysis, reaction of the AEC via opening of the β-lactam fused five-membered ring occurs to give one or more relatively hydrolytically stable species (Figs. 1B and ​and2).2). The nature of these species is central to SBLi inhibition and has been studied by crystallography (6–11) and ultraviolet-visible (UV/Vis) (10, 12) and Raman (6, 7, 9, 12–15) spectroscopy, as well as different types of mass spectrometry (MS) (10, 16–22).Open in a separate windowFig. 1.Sulfone derivatives of penicillins are potent clinically used mechanism-based inhibitors of SBLs. (A) Outline mechanism for penicillin hydrolysis as catalyzed by SBLs; reaction proceeds via an AEC, which is efficiently hydrolyzed. (B) Sulfone derivatives of penicillins are SBLi that react to give one or more hydrolytically stable complex(es), the nature of which was the focus of our work.Open in a separate windowFig. 2.Pathways for reactions of penam sulfones with SBLs. Following initial acyl–enzyme 2 formation the main transient inactivation pathway occurs via thiazolidine ring opening to give species 3-5 which are relatively stable to hydrolysis. Fragmentation of 3-5 can occur in rare cases and is promoted by acid to give 6-8 or heat to give 11. In rare cases fragmentation of 2-5 can result in irreversible inactivation of the SBL to give 9 and 10. Efficient hydrolysis of the β-lactam occurs to give a β-amino acid product 12, which in solution fragments to give 13-16. Our results imply biologically relevant inhibition involves 3-5, or equivalent mass species.The structures of tazobactam and sulbactam are closely related to those of the penicillins; they differ by lack of a C-6 side chain, functionalization of the pro-S methyl group (in case of tazobactam), and by oxidation of the thiazolidine to a sulfone. These differences result in a loss of useful antibacterial activity but a gain of potent SBL inhibition. Although the presence of sulfur in drugs is common [e.g., sulfonamide antibiotics (23)] and there is growing interest in covalently acting drugs (24, 25), sulfones are rare in drugs and, as far as we are aware, sulbactam and tazobactam are the only clinically approved sulfone-containing drugs working by covalent reaction with their targets (26–28).Since the clinical introduction of the pioneering SBLi, β-lactamases have evolved and SBLi use is increasingly compromised by extended spectrum β-lactamases (ESBLs) and inhibitor-resistant SBLs (29). Efforts have been made to develop new SBLi, including those with and without a β-lactam. The latter include diazabicyclooctanes (30) and cyclic boronates (31, 32). However, β-lactam–containing SBLi remain of most clinical importance. Among SBLi in clinical development, enmetazobactam (formerly AAI-101; Fig. 1) is of particular interest because it is a “simple” N-methylated derivative of the triazole ring of tazobactam (33). In combination with cefepime, enmetazobactam is reported to manifest substantially better antimicrobial properties against class A ESBL-producing strains than the commonly used piperacillin/tazobactam combination (20, 33, 34).We report studies on the mechanism of SBL inhibition by enmetazobactam using denaturing and nondenaturing (native) MS methods, NMR spectroscopy, and crystallography. The results led us to reevaluate the mechanisms of SBL inhibition by the clinically important sulfone-containing SBLi, i.e., tazobactam and sulbactam, and reveal limitations on the interpretation of MS studies concerning SBL inhibition.     

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson’s disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder caused by loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase). GCase degrades glycosphingolipids (GSLs), including glucosylceramides (GluCers), into glucose and ceramide, and GCase mutations result in the accumulation of GluCer in lysosomes of various tissues. Heterozygote carriers of the same loss-of-function GCase mutations are estimated to be at 5- to 10-fold higher risk for developing Parkinson’s disease (PD) or Lewy body dementia (1). In GD, significant variability exists in the clinical and pathological presentation, resulting in three main GD subtypes (2). Type 1 GD is characterized by visceral abnormalities, including enlarged liver and spleen and bone marrow dysfunction, leading to thrombocytopenia but without neurodegeneration and α-synuclein (α-syn) pathology (3). Types 2 and 3 demonstrate similar visceral symptoms but with additional extensive neuronal loss, α-syn pathology in the form of classical Lewy bodies, and neurological dysfunction (3, 4). As life expectancy of type 1 GD has increased because of enzyme replacement therapy, a higher percentage of patients develop PD symptoms with age (5), suggesting that aging could contribute to the penetrance of GBA1 mutations. The dramatic phenotypic heterogeneity suggests that GD is not a simple, monogenic disease but a complex disorder that is influenced by both genetic and nongenetic modifiers. Although the factors that contribute to clinical and pathological variability in GD are not known, genetic modifiers have been identified that associate with GD severity, including CLN8 and SCARB2 (6, 7). Within PD patients that harbor GBA1 mutations (GBA-PD), the search for genetic modifiers has shown that synergism may exist with the SNCA gene that encodes α-syn and CTSB that encodes lysosomal cathepsin B (8). Variants in lysosomal cathepsins could influence the severity of α-syn accumulation, since, under physiological or pathological conditions, α-syn can be degraded by the lysosome (9–11) and is a direct substrate of cathepsin B and L (12).An additional factor that may contribute to phenotypic variability in GD is the accumulation of specific GluCer subtypes with particular acyl chain lengths. GluCer and other GSLs exist as a family of lipid isoforms differentiated by the length of the N-acyl fatty acid moiety linked to the sphingoid base. GluCer chains range from C14 to C26 in the brain; however, C18 and C24:1 are the predominant species (13). Studies of neuronopathic GD (nGD) brain or mouse models showed intraneuronal accumulation of multiple GluCer species that correlated with neuroinflammation (14–19), and some cases demonstrate selective accumulation of long-chain GluCers in nGD (20). Our recent work in PD patient midbrain neurons showed that inhibition of wild-type (wt) GCase, caused by α-syn, resulted in the selective accumulation of long-chain-length GluCers, including C22 and C24:1, while C14, 16, and C18 were unchanged (21). Together, these data indicate that GluCer accumulation plays an important role in neurodegeneration induced by GBA1 mutations; however, the specific contributions of distinct GluCer species have not been examined.Here, we extend our studies on the role of GSLs in α-syn aggregation to further define conditions that are required to induce pathology and neurological dysfunction. We previously showed that α-syn exists as monomers and high–molecular weight (HMW) oligomers under physiological conditions in human midbrain cultures (22). In vitro, we found that GluCer mildly induced aggregation of α-syn monomers but primarily acted on physiological oligomers to convert them into toxic oligomers and fibrillar inclusions (22). α-syn accumulation can be prevented or reversed by reducing GSLs with GluCer synthase inhibitors (GCSi) in both GD and PD patient cultures, as well as in mouse models (22–24). While this work suggests a close relationship between GCase function and α-syn pathology, additional factors must exist that create a permissive environment for α-syn accumulation. Indeed, studies that used newborn mice or embryonic primary neuron cultures treated with the GCase inhibitor, conduritol beta epoxide (CBE), have shown no changes in α-syn despite reduced GCase activity (25–27). However, other studies that use matured neuron cultures, neuronal cell lines, or adult mice have shown that CBE dramatically induces α-syn aggregates (22, 28–31). We used an in vivo GD model and induced pluripotent stem cell (iPSC)–derived patient midbrain cultures to identify specific conditions that are required to induce α-syn pathology, providing possible explanations for the variable neurological penetrance in patients that harbor GBA1 mutations.     

Interlocking activities of DNA polymerase β in the base excision repair pathway

Base excision repair (BER) is a major cellular pathway for DNA damage repair. During BER, DNA polymerase β (Polβ) is hypothesized to first perform gap-filling DNA synthesis by its polymerase activity and then cleave a 5′-deoxyribose-5-phosphate (dRP) moiety via its dRP lyase activity. Through gel electrophoresis and kinetic analysis of partial BER reconstitution, we demonstrated that gap-filling DNA synthesis by the polymerase activity likely occurred after Schiff base formation but before β-elimination, the two chemical reactions catalyzed by the dRP lyase activity. The Schiff base formation and β-elimination intermediates were trapped by sodium borohydride reduction and identified by mass spectrometry and X-ray crystallography. Presteady-state kinetic analysis revealed that cross-linked Polβ (i.e., reduced Schiff base) exhibited a 17-fold higher polymerase efficiency than uncross-linked Polβ. Conventional and time-resolved X-ray crystallography of cross-linked Polβ visualized important intermediates for its dRP lyase and polymerase activities, leading to a modified chemical mechanism for the dRP lyase activity. The observed interlocking enzymatic activities of Polβ allow us to propose an altered mechanism for the BER pathway, at least under the conditions employed. Plausibly, the temporally coordinated activities at the two Polβ active sites may well be the reason why Polβ has both active sites embedded in a single polypeptide chain. This proposed pathway suggests a corrected facet of BER and DNA repair, and may enable alternative chemical strategies for therapeutic intervention, as Polβ dysfunction is a key element common to several disorders.

One of the major cellular pathways for repair of DNA damage is base excision repair (BER) (1–5). In this pathway (Scheme 1A), DNA lesions are removed by glycosylases (e.g., uracil by uracil–DNA glycosylase [UDG]) before the damaged DNA strand is incised by apurinic/apyrimidinic (AP) endonuclease, resulting in a single-nucleotide gap flanked by a 3′-OH and a 5′-deoxyribose-5-phosphate (dRP) moiety. Subsequently, DNA polymerase β (Polβ) is presumed to first catalyze gap-filling DNA synthesis through its DNA polymerase activity and then perform dRP cleavage via its dRP lyase activity, leaving a nicked DNA substrate for ligation by either Ligase III/XRCC1 or Ligase I (6–12).Open in a separate windowScheme 1.The BER pathway. (A) The BER pathway in the literature as cited in the Introduction. (B) Our proposed BER pathway.The dRP lyase active site resides within the 8-kDa N-terminal domain of Polβ, whereas the polymerase active site sits at the palm subdomain (SI Appendix, Fig. S1A) (7). Previously, Polβ has been shown to remove a dRP moiety through a Schiff base–mediated β-elimination reaction (13) rather than through hydrolysis (7–10, 14, 15). A Schiff base is generated following nucleophilic attack by the side chain of an active site lysine residue on the sugar C1′ atom of the dRP moiety (step 1 in Scheme 2). Whereas biochemical data suggest that K72 in human polymerase-β (hPolβ) acts as the active site nucleophile, conflicting evidence as well as a lack of supporting structural data have complicated understanding of the dRP cleavage mechanism (10, 14, 15). For instance, mutation of K72 to alanine does not fully abrogate the dRP lyase activity, suggesting that a different residue may support the nucleophilic attack on the C1′ (11). Furthermore, only limited conclusions can be drawn from the existing binary crystal structures of hPolβ bound to either a single-nucleotide gapped DNA substrate (hPolβ•DNA^P) (SI Appendix, Fig. S2B) containing only a 5′-phosphate, rather than a full dRP moiety, on the downstream primer (SI Appendix, Fig. S2 A, i) (16) or a nicked DNA substrate (hPolβ•DNA^THF) (SI Appendix, Fig. S2C) containing a 5′-dRP mimic (SI Appendix, Fig. S2 A, ii) (11). Due to the lack of the deoxyribose moiety in the structure of hPolβ•DNA^P, information about the dRP cleavage mechanism is lacking. On the other hand, in the structure of hPolβ•DNA^THF, the nonnatural dRP mimic was bound in a nonproductive docking site stabilized through the interaction between its 5′-phosphate and K68 (SI Appendix, Fig. S2C). This nonproductive site is distinct from the putative dRP lyase active site as the Nε atom of K72 is more than 10 Å from the dRP sugar C1′ (11). In fact, from this position, the dRP must rotate ∼120° around the 3′-phosphate to be in close-enough proximity to the Nε atom of K72 for nucleophilic attack to occur (SI Appendix, Fig. S2C). Furthermore, the active site residues responsible for stabilizing the reactive ring-opened aldehyde state of the dRP moiety and abstracting a proton from the ribose C2′ atom to facilitate β-elimination (Scheme 2) remain unidentified.Open in a separate windowScheme 2.Proposed chemical mechanism for the dRP lyase activity of hPolβ. Specific water molecules are denoted as X, Y, and Z.Biochemical studies of the processing of dRP moieties in yeast cell-free extract (17), steady-state kinetic studies of fully reconstituted human BER (4), and investigation of the numbers of endogenous AP sites in genomic DNA of rats and human tissue (5) all suggest that dRP cleavage is the rate-limiting step of the entire BER pathway. However, there is no experimental evidence to indicate that all potential steps associated with dRP cleavage by the lyase activity of Polβ (Scheme 2) occur after gap-filling DNA synthesis catalyzed by the polymerase activity. For example, if facile Schiff base formation occurs before and faster than nucleotide incorporation, the covalently linked Polβ–DNA intermediate, rather than the noncovalent binary complex Polβ•DNA, may catalyze gap-filling DNA synthesis. This possibility has never been investigated, and all previously published in vitro studies have used DNA substrates like either DNA^P (SI Appendix, Fig. S2 A, i) (18–24) or a gapped DNA substrate containing a dRP mimic (SI Appendix, Fig. S2 A, ii) (11, 25).Here, we generated a natural dRP moiety by using either UDG to process a nicked DNA substrate containing a 2′-deoxyuridine or UDG and apurinic/apyrimidinic endonuclease 1 (APE1) to initiate BER on a double-stranded DNA substrate containing a 2′-deoxyuridine. Addition of hPolβ, correct deoxynucleoside triphosphate (dNTP), and then, sodium borohydride (NaBH₄) to the dRP-containing DNA products allowed for the capture of a reduced Schiff base and a β-elimination intermediate produced via hPolβ-catalyzed dRP cleavage (Scheme 2). Through X-ray crystallographic, kinetic, and mass spectrometric (MS) analysis of these cross-linked hPolβ complexes, we envisioned a detailed chemical mechanism for the dRP lyase activity of hPolβ. In addition, we utilized presteady-state kinetic methods to evaluate the impact of the reduced Schiff base intermediate on the efficiency and fidelity of gap-filling DNA synthesis by the polymerase activity of hPolβ. Finally, we employed time-resolved X-ray crystallography to structurally characterize intermediates of gap-filling DNA synthesis by cross-linked hPolβ. Based on several lines of experimental evidence, we proposed a modified BER pathway (Scheme 1B), which posits an interlocking mechanism in which gap-filling DNA synthesis by the polymerase activity occurs between Schiff base formation and β-elimination, the two steps catalyzed by the lyase activity.     

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

The cell envelopes of gram-negative bacteria, mitochondria, and chloroplasts all contain an outer membrane (OM) consisting of integral transmembrane proteins that assume a β-barrel conformation (1, 2). In gram-negative bacteria such as Escherichia coli, β-barrel outer membrane proteins (OMPs) contribute to the selective permeability of the OM, protecting the cell from harmful molecules while still allowing for the uptake of nutrients (3). Structurally and functionally diverse OMPs serve a number of roles critical to cell viability, namely the selective passage of small molecules, efflux of toxins, insertion of lipopolysaccharide (LPS) onto the cell surface, and assembly of OMPs themselves (1, 4). Reflective of their importance in maintaining cellular integrity, defects in OMP biogenesis confer sensitivity to a wide array of toxic molecules including detergents, bile salts, and most importantly, antibiotics (5, 6). As such, considerable efforts have been made to identify agents that inhibit essential cellular processes performed by OMPs (7–12), with hopes of hastening the development of novel therapeutics to combat the ever-growing threat of antibiotic-resistant infections caused by gram-negative microbes (13, 14).Ensuring efficient OMP biogenesis is a particularly challenging cellular feat. Newly synthesized OMPs must traverse the aqueous, oxidizing periplasm in an unfolded state, avoid self-aggregation, and subsequently complete proper assembly, all in an environment devoid of cellular energy such as adenosine triphosphate (15). A multitude of molecular chaperones and proteases function to overcome this challenge by minimizing unfolded OMP accumulation and facilitating OMP transport to the OM assembly machinery (16). Although more than a dozen chaperones and proteases with clear implications in OMP biogenesis have been identified (16–18), the most well-characterized and predominant proteins in E. coli are the chaperones SurA and Skp, as well as the chaperone protease DegP. Numerous genetic, biochemical, and proteomic studies have demonstrated that SurA is the primary periplasmic chaperone that facilitates transport of the bulk mass of OMP substrates to the OM (19–24). Skp and DegP, on the other hand, comprise a secondary, partially redundant OMP biogenesis pathway that primarily serves to minimize accumulation of unfolded OMPs, either by rescuing their assembly or promoting their degradation (19, 20).Notably, Skp binds unfolded OMPs with dissociation constants in the low nanomolar range (25, 26), exceeding the binding affinities of either SurA or DegP (27–29), to form highly stable Skp–OMP complexes that display lifetimes on the order of hours (30). Given the substantial stability of Skp–OMP complexes, the precise mechanism of OMP release from Skp remains poorly understood. The rapid conformational dynamics of OMPs bound within the Skp cavity have been proposed to enable local substrate release that is ultimately driven by the recognition and folding of OMPs by the OM assembly machinery (30), thus coupling client release from Skp to the thermodynamic stability provided by OMP integration into a membrane (31). Indeed, substrate release and folding of OMPs from Skp–OMP complexes is enabled in vitro by incubation with OM folding machinery–containing liposomes (28, 32), demonstrating that Skp can facilitate productive OMP assembly. This mechanism of folding-driven substrate release has been similarly observed in genetic and biochemical studies indicating that Skp is capable of directly inserting OMPs into lipid bilayers in vitro (33), as well as the inner membrane in vivo (34), without assistance from the OM assembly machinery.Whether OMPs are capable of being removed from Skp within physiological timescales in the absence of coupled folding, however, is not entirely clear. Under conditions of periplasmic stress, in which the burden of unfolded OMPs exceeds the rate at which they can be assembled, the activities of both Skp and DegP become crucial (19, 20, 24, 35). Given that Skp not only binds substrates with a higher affinity than DegP (29) but also does so several orders of magnitude faster (36), how unfolded OMPs are transferred from Skp to DegP for degradation under stress conditions is not obvious. Indeed, direct transfer of an OMP from Skp to DegP has yet to be demonstrated, and intriguingly, the formation of Skp–DegP–OMP ternary complexes has been reported in such experiments (29, 36).Folding and insertion of nascent OMPs into the OM is catalyzed by the heteropentameric β-barrel assembly machine (Bam) complex, consisting of the BamA β-barrel and four accessory lipoproteins, BamBCDE (37, 38). Recent biochemical and structural studies have provided a relatively clear current model for the mechanism of β-barrel assembly. Following substrate recruitment to BamD (39), BamA catalyzes the sequential addition of β-hairpins in a C-to-N-terminal manner (40), with early folding occurring within the interior of the BamA barrel (41). Folding proceeds until membrane integration occurs, and subsequent stepwise hydrogen-bond formation between N and C substrate termini facilitates barrel closure and substrate release into the membrane (40).One outstanding question concerns the fate of OMP substrates that have stalled while folding on the Bam complex. Protein misfolding in the periplasm, translational error, or impaired Bam complex function can result in substrates arresting on the assembly machinery, a condition that can ultimately be lethal if left unchecked (42–44). Until recently, investigations of stalled OMP substrates have been largely impeded by a lack of structurally defined folding intermediates and the absence of an established general mechanism of OMP assembly. Several studies to date have utilized mutant alleles of the large β-barrel LptD to probe Bam complex assembly (39, 41, 45, 46), and multiple proteases that degrade assembly-compromised LptD within distinct stages of its folding regime have been identified (46, 47). It is unclear, however, whether these stringent quality control mechanisms monitoring assembly of LptD are exerted on all β-barrel substrates or whether LptD represents a unique case given its remarkably complex folding trajectory (48). Given that OMP assembly by the Bam complex has evolved to be incredibly efficient—so efficient that unfolded OMPs cannot be detected at steady state—it stands to reason that quality control mechanisms ensuring the efficient assembly of all β-barrel substrates exist. Recently, it has been shown that extracellular loop deletions within the C-terminal half of the BamA β-barrel cause early folding defects and thus render stalled BamA susceptible to proteolysis by DegP (40). How DegP actively disengages a partially folded, stalled substrate from its folding on BamA, given the relatively weak and slow nature of DegP binding, is not obvious.Here, we have utilized an assembly-defective variant of a slow-folding β-barrel OMP to investigate the fate of substrates that engage the OM assembly machinery but otherwise fail to undergo efficient folding and membrane integration. We identify a specific role for the periplasmic chaperone Skp in facilitating the degradation of defective OMP substrates by the protease DegP, thus imposing an active quality control mechanism that serves to remove assembly-compromised substrates from the Bam complex. Strikingly, we find that Skp is degraded alongside its bound substrate by DegP, thereby functioning as a sacrificial adaptor protein. By evaluating the requirement for Skp in degradation of a series of sequentially stalled β-barrel substrates, we find that Skp is only required to degrade substrates that have initiated folding on the Bam complex. Thus, β-barrel OMPs that have stalled during assembly specifically require Skp for their removal from the Bam complex and subsequent degradation by DegP. We conclude that Skp acts to ensure efficient β-barrel assembly by facilitating both the direct removal and degradation of stalled substrates from the Bam complex.     

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell–killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy.

Macrophages are innate immune cells that show phenotypic heterogeneity and functional diversity; and they play key roles in development, tissue homeostasis and repair, and in cancer, as well as in defense against pathogens (1–3). In the tumor microenvironment (TME), macrophages are exposed to a variety of stimuli, including cell–cell contact, hypoxia, as well as soluble and insoluble factors such as cytokines, chemokines, metabolites, and extracellular matrix components (2, 4). These environmental cues promote the acquisition by macrophages of protumorigenic phenotypes that facilitate tumor development, progression, and metastasis as well as suppress antitumor immune responses (2, 4). A high density of macrophages within tumor tissue is associated with poor prognosis in patients with various types of cancer, including that of the bladder or breast (5–7). Depletion of macrophages in the TME or the reprogramming of these cells to acquire antitumorigenic phenotypes has been shown to ameliorate the immunosuppressive condition and result in a reduction in tumor burden in both preclinical and clinical studies (2, 4, 8, 9). Macrophages within the TME have therefore attracted much attention as a potential therapeutic target for cancer immunotherapy.Signal regulatory protein α (SIRPα) is a transmembrane protein that possesses one NH₂-terminal immunoglobulin (Ig)-V–like and two Ig-C domains in its extracellular region, as well as immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic region (10, 11). The extracellular region of SIRPα interacts with that of CD47, another member of the Ig superfamily of proteins, with this interaction constituting a means of cell–cell communication. The expression of SIRPα in hematopoietic cells is restricted to the myeloid compartment—including macrophages, neutrophils, and dendritic cells (DCs)—whereas CD47 is expressed in most normal cell types as well as cancer cells (12, 13). The interaction of SIRPα on macrophages with CD47 on antibody (Ab)-opsonized viable cells such as blood cells or cancer cells prevents phagocytosis of the latter cells by the former (13–15), with this negative regulation of macrophages being thought to be mediated by SHP1, a protein tyrosine phosphatase that binds to the cytoplasmic region of SIRPα (14). Indeed, blockade of the CD47–SIRPα interaction by Abs to either SIRPα or CD47, in combination with a tumor-targeting Ab such as rituximab (anti-CD20), was found to enhance the Ab-dependent cellular phagocytosis (ADCP) activity of macrophages for cancer cells that do not express SIRPα, resulting in marked suppression of tumor formation in mice (15–19). Targeting of SIRPα in combination with a tumor-targeting Ab therefore provides a potential approach to cancer immunotherapy dependent on enhancement of the ADCP activity of macrophages for cancer cells. In contrast, the effect of Abs to SIRPα in the absence of a tumor-targeting Ab on the phagocytosis by macrophages of, as well as on tumor formation by, cancer cells that do not express SIRPα was minimal or limited.We have now further examined the antitumor efficacy of a monoclonal Ab (mAb) to mouse SIRPα (MY-1) (20) in immunocompetent mice transplanted subcutaneously with several types of murine cancer cells that do not express SIRPα. This Ab prevents the binding of mouse CD47 to SIRPα and cross-reacts with mouse SIRPβ1 (15). We found that monotherapy with MY-1 efficiently attenuated the growth of tumors formed by bladder or mammary cancer cells. In addition, MY-1 markedly promoted the induction of antitumorigenic macrophages able to target these cancer cells. Furthermore, our results suggest that SIRPβ1 on macrophages likely participated in the antitumorigenic effect of MY-1.     

Mechanistic basis for receptor-mediated pathological α-synuclein fibril cell-to-cell transmission in Parkinson's disease

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson’s disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

Aggregation and the spread of amyloid proteins, such as α-synuclein (α-syn), amyloid-β, Tau, and TDP43, are critical events in the pathogenesis of neurodegenerative disorders, including Parkinson''s disease (PD), Alzheimer’s disease, and amyotrophic lateral sclerosis, respectively (1, 2). As the hallmark of PD and other α-synucleinopathies, α-syn aggregation spreads in a prion-like progressive and stepwise manner both within the brain and from other organs to the brain during disease progression (3–7). Pathological α-syn aggregation can template monomeric α-syn to aggregate and participate in disease pathogenesis. Pathological α-syn inclusion can spread in the grafted neurons of PD patients (4, 8). Brain extracts from patients with multiple system atrophy can transmit neurodegeneration to genetically engineered mice (9). A single administration of α-syn preformed fibrils (PFFs) in mouse brains can recapitulate the pathological phenotypes of α-synucleinopathies (10–13).Selected cell surface proteins, such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1), have been found to serve as receptors for α-syn PFF internalization and transmission (10, 14, 15). Intriguingly, these receptors preferentially recognize α-syn PFFs rather than the monomer (10). The α-syn monomer is intrinsically disordered and forms α-helical conformation upon membrane binding as involved in synaptic vesicle trafficking (16–19). Cryogenic electron microscopic (cryo-EM) structures of full-length α-syn amyloid fibrils show that the central region of α-syn, approximately covering residues 37 to 99, is involved in the formation of a cross-β fibril core (termed as FC region), while the remaining N and C termini remain flexible (20–24). Despite the recent successes in the structural determination of α-syn amyloid fibrils, considerable challenges remain in linking the structural information to α-syn pathology. The structural basis underlying α-syn transmission, specifically the interplay between α-syn PFFs and receptors, is unknown. It also remains unclear how receptors, for example, LAG3 and APLP1, selectively recognize α-syn PFFs over monomers, nor do we know the role of posttranslational modification of α-syn in this process.In this work, we combined multiple biophysical, cellular, and in vivo approaches to reveal the structural basis underlying the receptor binding of α-syn amyloid fibrils during cell-to-cell transmission. We found that the D1 domain of LAG3 utilizes a positively charged surface to capture the acidic C terminus of α-syn, which is exposed and concentrated on the surface of α-syn fibrils. In contrast, α-syn monomers adopt a self-shielded conformation to impede the exposure of the C terminus. Phosphorylation at serine 129 (pS129) of α-syn, a pathological biomarker in PD (25–27), significantly enhances the binding of α-syn PFFs to LAG3 and APLP1 and promotes the cell-to-cell transmission in vitro and in vivo. Our work provides the structural basis for the receptor-mediated neuronal internalization and transmission of α-syn fibrils and suggests that the C terminus, specifically residues 118 to 140, is a pathological epitope of α-syn for receptor binding and thus may serve as a promising target for the therapeutic drug development to block PD progression.     

β-Arrestin–biased signaling mediates memory reconsolidation

A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of β₁-adrenergic β-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. β-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain β-adrenergic Gs protein- and β-arrestin–dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the β-arrestin/ERK signaling by the biased β-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of β-arrestin2 in the entorhinal cortex of β-arrestin 2–deficient mice rescues β₁-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that β-arrestin–biased signaling regulates memory reconsolidation and reveal the potential for β-arrestin–biased ligands in the treatment of memory-related disorders.Alongside classical G protein pathways, activation of G protein-coupled receptors (GPCRs) stimulates β-arrestin–dependent signaling, leading to ERK phosphorylation and other downstream events (1, 2). Biased agonists, which induce functionally selective or biased receptor states and, thus, selectively activate one of the signaling pathways, have recently been identified for several GPCRs (3). Biased receptor agonism offers theoretical guidance for the discovery of a new generation of GPCR-targeted drugs with greater efficacy but fewer adverse effects. However, the lack of knowledge about the signaling pathways specifically eliciting a beneficial effect is a major obstacle in the understanding of disease mechanisms and the development of biased drugs targeting most GPCRs, especially those expressed in the central nervous system (CNS) with psychiatric importance.Besides their important roles in the cardiovascular and pulmonary systems, β-adrenergic receptors (β-ARs) are critically involved in CNS functions such as arousal, cognition, and stress-related behaviors (4, 5). β-Adrenergic neuronal signaling is important for neuroplasticity, including long-term potentiation (6) and memory formation (7). Accumulating cell biological evidence suggests that β-ARs also signal via G protein-independent, β-arrestin–dependent pathways (8–10). However, functions of β-AR in the CNS have been primarily ascribed to their classical role of stimulating Gs protein. The differential neurophysiological consequences for the G protein- and β-arrestin–dependent pathways, if any, have not been delineated.A longstanding hypothesis posits that a β-AR/Gs/protein kinase A (PKA) signaling pathway mediates memory reconsolidation (11–13), a process that strengthens, updates, or erases a previously acquired memory after recall (memory reactivation). This hypothesis is largely based on observations that β-ARs and molecules in the classical GPCR signaling pathway—such as cAMP (cAMP), PKA, and cAMP response element-binding protein (CREB)—are required for reconsolidation, which was determined by using receptor antagonists, kinase inhibitors, or gene knockout mice (11, 14, 15). Most of these molecules are also required for basal neural activity or plasticity, and there has been no direct evidence demonstrating that the function of β-ARs in reconsolidation is mediated by G protein/PKA or other signaling pathway (12). In the current study, we tested the potential involvement of G protein/cAMP/PKA-dependent pathway versus β-arrestin–dependent signaling in memory reconsolidation by using object recognition paradigm.     

Ceramide accumulation induces mitophagy and impairs β-oxidation in PINK1 deficiency

Energy production via the mitochondrial electron transport chain (ETC) and mitophagy are two important processes affected in Parkinson’s disease (PD). Interestingly, PINK1, mutations of which cause early-onset PD, plays a key role in both processes, suggesting that these two mechanisms are connected. However, the converging link of both pathways currently remains enigmatic. Recent findings demonstrated that lipid aggregation, along with defective mitochondria, is present in postmortem brains of PD patients. In addition, an increasing body of evidence shows that sphingolipids, including ceramide, are altered in PD, supporting the importance of lipids in the pathophysiology of PD. Here, we identified ceramide to play a crucial role in PINK1-related PD that was previously linked almost exclusively to mitochondrial dysfunction. We found ceramide to accumulate in mitochondria and to negatively affect mitochondrial function, most notably the ETC. Lowering ceramide levels improved mitochondrial phenotypes in pink1-mutant flies and PINK1-deficient patient-derived fibroblasts, showing that the effects of ceramide are evolutionarily conserved. In addition, ceramide accumulation provoked ceramide-induced mitophagy upon PINK1 deficiency. As a result of the ceramide accumulation, β-oxidation in PINK1 mutants was decreased, which was rescued by lowering ceramide levels. Furthermore, stimulation of β-oxidation was sufficient to rescue PINK1-deficient phenotypes. In conclusion, we discovered a cellular mechanism resulting from PD-causing loss of PINK1 and found a protective role of β-oxidation in ETC dysfunction, thus linking lipids and mitochondria in the pathophysiology of PINK1-related PD. Furthermore, our data nominate β-oxidation and ceramide as therapeutic targets for PD.

Loss of PINK1 function causes autosomal recessive early-onset Parkinson’s disease (PD). Most patients present with bradykinesia, rigidity, resting tremor, and dyskinesia and are responsive to dopamine replacement therapy (1). On the cellular level, PINK1 disease mutations result in impaired energy metabolism and a variety of mitochondrial defects that can partially be alleviated by stimulation of energy metabolism (2–4). Intriguingly, abnormal mitochondrial morphology, along with lipid aggregates, was recently discovered to be present in Lewy bodies of postmortem PD patients’ brains (5), challenging the previously held notion of alpha-synuclein being the almost exclusive neuropathological correlate. This finding confirms the involvement of mitochondrial dysfunction in PD and additionally suggests a critical role of lipids in the pathogenesis of PD.PINK1 is important for the phosphorylation of the Complex I subunit NdufA10 resulting in efficient Complex I and electron transport chain (ETC) activity (6, 7). This function is evolutionarily conserved between Drosophila and humans. Hence, in both flies and humans, loss of PINK1 results in an impaired ETC, reduced ATP levels, and defective mitochondrial morphology (6, 8, 9), all of which are ubiquitously observed in the fly already at the early larval stage. Furthermore, alongside Parkin, PINK1 plays a crucial role in mitophagy to remove defective mitochondria that appears to be defective in an age-dependent fashion (10–13). Pink1-mutant Drosophila melanogaster additionally show thorax muscle degeneration and defective flying ability (8, 9). These latter defects, together with impaired mitochondrial morphology, can be rescued by expressing the fission-promoting protein Drp1 (14). However, increased fission does not improve ETC-related defects (15). Furthermore, stimulation or facilitation of the ETC rescues ETC-related phenotypes in pink1-mutant Drosophila, including ATP levels and mitochondrial morphology (3, 4, 7, 15, 16). These data collectively suggest two parallel mechanisms that converge on a shared common pathway leading to the development of PD. However, the link between these two pathways has yet to be resolved.Recently, disrupted lipid homeostasis has garnered increasing attention in PD (16–18). Furthermore, ceramide, the basic sphingolipid, is altered in several PD models and has been implicated in PD-related alpha-synuclein toxicity (17–20). Interestingly, ceramide induces mitophagy that is facilitated by Drp1 (21). Furthermore, pathogenic variants in Glucocerebrosidase (GCase), an enzyme involved in ceramide synthesis, are known to be the most common risk factor for PD (22, 23). However, the exact mechanism remains enigmatic. We found increased ceramide levels in isolated mitochondria of Pink1^−/− knockout (KO) mouse embryonic fibroblasts (MEFs) (16) and Pink1-deficient flies. Increased ceramide levels are detrimental for proper ETC function (24). Hence, we hypothesize that ceramide accumulation in PINK1 deficiency affects ETC function and mitophagy and constitutes the missing link between these two important processes affected in PD.     

The N terminus of α-synuclein dictates fibril formation

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson’s disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41–140). This behavior can be explained by the unique fibril structure that is adopted by 41–140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Amyloid formation of α-synuclein (α-syn) is a pathological feature of Parkinson’s disease (PD), multiple-system atrophy (MSA), and dementia with Lewy bodies (1, 2). An abundant presynaptic protein (3), α-syn is 140 amino acids in length with a putative biological function in aiding the exocytosis of synaptic vesicles (4–6), in which the first 89 N-terminal residues fold into a helical structure upon membrane association (7). In its disease-associated, aggregated amyloid state, residues 37 through 97 adopt β-sheet structure (8), which overlaps with the lipid-binding domain. Notably, both N- and C-terminal α-syn truncations are associated with PD (9). So far, N-terminally truncated (ΔN) α-syn variants 5‒140, 39‒140, 65‒140, 66‒140, 68‒140, and 71‒140 and C-terminally truncated (ΔC) α-syn variants, 1‒101, 1‒103, 1‒115, 1‒122, 1‒124, 1‒135, and 1‒139 have been found in brains of PD patients (10–12).α-Syn truncations originate from incomplete degradation, which has been attributed to various cytosolic (13–15) and lysosomal proteases (16, 17). In fact, ∼60% of the abovementioned truncations can be assigned to cleavages by lysosomal asparagine endopeptidase (AEP), cathepsin (Cts) D, CtsB, and CtsL (15–17). Removal of the C terminus (residues 104–140) is shown to accelerate fibril formation both in vitro and in vivo (18–25). On the other hand, perplexing behaviors of ΔN-variants have been documented; while deleting the first 20 residues has minimal perturbation, the removal of either the first 10 or 30 residues slows aggregation kinetics (26). Nevertheless, the influence of N-terminal residues on α-syn aggregation has been shown by both insertion [tandem repeat of residues 9–30 (27)] and deletion [Δ36–42 (28) and Δ52–55 (29)] mutants, in which fibril formation can be completely impeded.Recently, structure determination by cryogenic electron microscopy (cryo-EM) has revealed fibril structures for full-length α-syn (1–140) (24, 30–32), C-terminal truncations (24, 33), phosphorylated Y39 (34), and PD-related mutants, E46K (35, 36), H50Q (37), and A53T (38). One striking feature of these fibrils is the eclectic mix of structures, often termed as fibril polymorphism. In fact, it was recently shown that different conformational strains of α-syn fibrils are present in PD and MSA patients (39, 40). The outstanding question still remains as to how the same polypeptide chain can produce such a vast number of polymorphic structures. While there are significant structural differences, some features of α-syn fibrils are conserved. All fibrils are formed from a twisting pair of protofilaments with the exception of a H50Q polymorph, which is composed of a single filament. A kernel motif of a bent β-arch appears in all structures. Also, at least one inter- or intramolecular salt bridge between a Lys and Glu is revealed in each structure (24, 30–38, 40), which is not surprising given that there are numerous possibilities for salt bridges between the 14 Lys, 8 Glu, and 2 Asp residues located throughout the first 100 residues in the sequence (Fig. 1A and SI Appendix, Fig. S1). Generally, residues between 37 and 97 constitute the fibril core with a few exceptions that involve additional residues in the N terminus, which include phosphorylated Y39 fibrils with an extended core of 1–100 (34) and two polymorphs of 1–140 showing interactions of N-terminal β-strands (residues 14–24) (30). Fibrils derived from brains of MSA patients also indicate additional involvement of the N-terminal region extending to residue 14 (40). Due to the contribution of N-terminal residues in these structures and the fact that C-terminal truncations resulted in modest conformational changes, we hypothesize that N-terminal residues play a greater role in influencing fibril structure.Open in a separate windowFig. 1.Aggregation of ΔN-α-syns. (A) Schematic representation of α-syn primary sequence (residues 1–140), showing basic (blue) and acidic (red) residues. Underlined regions correspond to truncations used in this study: 14‒140 (blue), 36‒140 (magenta), and 41‒140 (green). (B and C) Comparison of aggregation kinetics monitored by ThT fluorescence at 37 °C. [α-Syn] = 35 µM (B) and 70 µM (C) with [ThT] = 10 µM in 20 mM NaPi, 140 mM NaCl, pH 7.4. The solid line and shaded region represent the mean and SD, respectively (n ≥ 4). Representative TEM images of (D) 1‒140, (E) 14‒140, (F) 36‒140, and (G) 41‒140 were taken at 35 µM. Different fibril polymorphs observed are noted. Additional fields of view are shown in SI Appendix, Figs. S3–S5.Here, we sought to understand the role of the N terminus in α-syn fibril formation by removing different N-terminal residues and evaluating their effects on aggregation kinetics, fibril structure, and propagation. Three ∆N-terminal constructs (14‒140, 36‒140, and 41‒140) have been examined, in which the first 13-, 35-, and 40-residues in the N terminus were deleted (Fig. 1A). We specifically chose these sites based on the locations of native Gly residues, which allows us to generate native sequences (i.e., no overhang) upon Tobacco Etch Virus (TEV) protease cleavage of the hexahistidine affinity tag, which facilitates facile protein purification. All three ∆N-α-syn exhibited different aggregation kinetics and distinct fibril ultrastructural features as determined by thioflavin-T (ThT) fluorescence and transmission electron microscopy (TEM), respectively. In cross-seeding experiments, both fibrillar 36‒140 and 41‒140 did not seed the full-length (1‒140) protein, while 14‒140 fared better but less efficient than self-seeding, supportive of the significant impact of removing N-terminal residues in fibril structure. The reverse reaction involving full-length seeds showed that fibril formation of 14‒140 and 36‒140 but not 41‒140 could be accelerated. This observation is explained by the fibril structure adopted by 41–140, which was determined by cryo-EM to an overall resolution of 3.2 Å. Unlike any currently known α-syn structure, the amyloid core is formed by two asymmetric protomers with different amino acid chain lengths, adopting an extended β-hairpin (E61‒D98) and the bent β-arch kernel (E46‒K96) with a large nonpolar interfilament interface (442 Ų) stabilized by an intermolecular salt bridge between K80 and E83. Collectively, these results establish the important role of N-terminal residues in fibril formation and structure.     

Membrane association of importin α facilitates viral entry into salivary gland cells of vector insects

The importin α family belongs to the conserved nuclear transport pathway in eukaryotes. However, the biological functions of importin α in the plasma membrane are still elusive. Here, we report that importin α, as a plasma membrane–associated protein, is exploited by the rice stripe virus (RSV) to enter vector insect cells, especially salivary gland cells. When the expression of three importin α genes was simultaneously knocked down, few virions entered the salivary glands of the small brown planthopper, Laodelphax striatellus. Through hemocoel inoculation of virions, only importin α2 was found to efficiently regulate viral entry into insect salivary-gland cells. Importin α2 bound the nucleocapsid protein of RSV with a relatively high affinity through its importin β–binding (IBB) domain, with a dissociation constant K_D of 9.1 μM. Furthermore, importin α2 and its IBB domain showed a distinct distribution in the plasma membrane through binding to heparin in heparan sulfate proteoglycan. When the expression of importin α2 was knocked down in viruliferous planthoppers or in nonviruliferous planthoppers before they acquired virions, the viral transmission efficiency of the vector insects in terms of the viral amount and disease incidence in rice was dramatically decreased. These findings not only reveal the specific function of the importin α family in the plasma membrane utilized by viruses, but also provide a promising target gene in vector insects for manipulation to efficiently control outbreaks of rice stripe disease.

The importin α family belongs to the conserved importin α/β nuclear transport pathway in eukaryotes (1–3). It is well known that the importin α family plays an indispensable role in transporting proteins from the cytoplasm to the nucleus, with diverse functions in gene regulation, cell physiology, and differentiation (1, 4, 5). In addition to nucleocytoplasmic transport, some members of the importin α family localize to the plasma membrane with palmitoylation modification or through binding to heparin in heparan sulfate proteoglycan (HSPG) (6–8). Increased importin α levels in the plasma membrane are concomitant with decreased importin α levels in the cytoplasm, which affect the nuclear import of cargos and regulates intracellular scaling (7). However, the function of the importin α family in the plasma membrane is still elusive.Many plant viruses are transmitted by vector insects in a persistent, circulative mode, which is characterized by systemic invasion of diverse tissues prior to entering salivary glands and release in saliva (9–13). The salivary glands are the last barriers for viral transmission to overcome (14–18). Unfortunately, the molecular mechanisms underlying viral entry into salivary-gland cells are not well known. The rice stripe virus (RSV) is a typical persistent, circulative plant virus and is capable of proliferating in the midgut epithelial cells and of being efficiently transmitted by the vector insect small brown planthopper (Laodelphax striatellus) (19). This virus causes one of the most destructive rice stripe diseases, showing an incidence of up to 80% and causing yield losses of 30 to 40% in the rice fields of Asian countries (20). RSV is a nonenveloped negative-strand RNA virus of the Tenuivirus genus (21, 22). The genome of RSV contains four single-stranded RNA segments, each of which is encapsidated by a viral nucleocapsid protein (NP). In addition to the NP, RSV encodes an RNA-dependent RNA polymerase and five nonstructural proteins (NS2, NSvc2, NS3, SP, and NSvc4) (23–25).In our recent work, we found that three importin α proteins, importin α1 (GenBank registration number {"type":"entrez-nucleotide","attrs":{"text":"MT036051","term_id":"2078901399","term_text":"MT036051"}}MT036051), α2 ({"type":"entrez-nucleotide","attrs":{"text":"MT036050","term_id":"2078901365","term_text":"MT036050"}}MT036050), and α3 ({"type":"entrez-nucleotide","attrs":{"text":"MT036052","term_id":"2078901435","term_text":"MT036052"}}MT036052), of the small brown planthopper participate in the nuclear entry of RSV through direct interactions with RSV NPs, triggering an antiviral caspase-dependent apoptotic reaction (26). Knockdown of the expression of all the three importin α genes retarded the nuclear entry of RSV and led to an increase in viral load in planthoppers (26). However, we did not determine the influence on viral transmission. In the present study, we surprisingly found that one of the importin α proteins, importin α2, is associated with the plasma membrane and efficiently facilitates viral entry into insect salivary glands and subsequent viral transmission.     

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Aberrant aggregation of α-synuclein (α-syn) into amyloid fibrils and subsequent accumulation into intracellular inclusions is a hallmark of neurodegenerative disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy (1–3). In these diseases, soluble α-syn monomers misfold and self-assemble, forming small oligomeric species that retain the highly disordered structure of the monomeric state (4). These species are rather unstable and can undergo structural rearrangements, including a gain in β-sheet structure that generates more stable species (4, 5). β-structured oligomers can grow further through monomer addition or self-association, finally giving rise to well-defined amyloid fibrils (4–6). Despite the controversial evidence about the relationship between the different species that populate the aggregation process and cellular toxicity, the prevalent view is that both intermediate oligomers and small fibrils are neurotoxic (7). Due to their abnormal interactions with cellular components, certain types of oligomers are key pathogenic agents in the development of the disease (8–10). In particular, they can disrupt membranes, induce oxidative stress, dysregulate calcium homeostasis, cause mitochondria dysfunction, or impair the proteasome system (11). Furthermore, α-syn oligomers have been implicated in the spreading of the disease, as these aggregates can be transmitted between cells (12, 13). Small fibrils have also been related to intercellular spreading and propagation of neurodegeneration (14–18). In contrast, large amyloid aggregates are believed to be relatively inert, as their highly ordered packing and slow diffusion reduces undesired interactions with cellular components. Even so, large aggregates can generate intermediate species that contribute to cytotoxicity through secondary processes such as fragmentation or nucleation on the aggregate surface (19, 20).To counteract the toxic effect of protein aggregates, cells have evolved a sophisticated protein homeostasis network that coordinates protein synthesis, folding, disaggregation and degradation (21). This network is composed of the translational machinery, molecular chaperones and cochaperones, the ubiquitin-proteasome system, and the autophagy machinery. The way this network tackles amyloid aggregates remains poorly understood. It has been previously reported that the constitutive human Hsp70 (Hsc70) in collaboration with its Hsp40 cochaperone (Hdj1 or DnaJB1) slowly disassembles preformed α-syn fibrils (22). This activity was further stimulated by adding the NEF Hsp110 (Apg2). HspB5, a small heat shock protein also known as αB-crystallin, potentiated α-syn fibril disassembly by the ternary chaperone mixture. Although this chaperone combination was able to disaggregate fibrils, they did it in a timescale of weeks through a depolymerization process. Only when Hsp104, a yeast representative of the Hsp100 family able of fragmenting fibrils, was added to the mixture, disassembly occurred within hours (22). The lack of Hsp104 homologs in metazoans questioned whether this activity was physiologically relevant in humans. A later study revealed that a chaperone complex composed solely of members of the Hsp70, Hsp40, and Hsp110 families was able to efficiently reverse α-syn amyloid fibrils through both fragmentation and depolymerization, generating smaller fibrils, oligomers, and, ultimately, monomers (23). Despite the importance of this emerging disaggregase functionality, its mechanism of action remains largely unknown. Recently, the same chaperone mixture has been reported to also disaggregate tau and Htt fibrils (24–26), pointing to this Hsp70-based machinery as a potential human amyloid disaggregase.The two-fold aim of this work is, firstly, to test whether human disaggregase remodels with the same efficiency the different aggregates that populate the complex process of amyloid formation and, secondly, to shed light on the key mechanisms involved in the disassembly of amyloids. We show that the human disaggregase system disassembles toxic oligomers and short fibrils much better than large, less toxic fibrils, and that it does so by an enhanced destabilization of the small aggregated forms. Explicitly, fibril disassembly involves destabilization of the fibril ends and unzipping of the protofilaments, which allow depolymerization. The fast propagation of protofilament depolymerization toward the opposite fibril end is consistent with entropic pulling forces exerted by Hsc70 upon binding the fibril surface.