Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOS^cat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOS^cat engaging KRas. We also find KRas^G13D exhibits high affinity for SOS^cat and is a potent allosteric modulator of its activity. A structure of the KRas^G13D•SOS^cat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRas^G13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRas^G13D•SOS^cat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Ras, a member of the small G-protein family, represents important signaling molecules with diverse cellular roles, such as cell differentiation and proliferation (1–4). Different isoforms of Ras (HRas, KRas, and NRas) have high overall sequence identity and are the most commonly mutated of all discovered oncogenes (5, 6). Of the three Ras isoforms, KRas is the most frequently mutated isoform in cancers, such as pancreatic cancer (70–90%), colon cancer (30–50%), and lung cancer (20–30%) (7, 8). Ras proteins regulate cell signaling pathways by cycling between inactive, GDP-bound, and active, GTP-bound states that is accompanied by remodeling of three key regions within Ras: p-loop (residues 10–17), switch I (residues 30–38), and switch II (residues 60–76) (9–11).As Ras proteins possess slow guanine nucleotide exchange rates, their activation is regulated by guanine nucleotide exchange factors (GEFs) that reload Ras with GTP (12, 13). The multidomain protein, Son of Sevenless (SOS), is a GEF with the cdc25 and Ras exchanger motif domains representing the minimal, functionally competent unit, termed SOS^cat (14). Structural studies have revealed two Ras binding sites to SOS^cat, leading to the discovery that binding of Ras–GTP at the distal (or allosteric) site allosterically modulates SOS^cat activity, which markedly increases the nucleotide exchange rate at the active site (14, 15). In addition, the degree of this allosteric modulation greatly depends on the nucleotide-bound state of Ras (14, 16, 17). Moreover, SOS is conformationally dynamic and binding of Ras at the allosteric site appears to shift the population to active conformation(s) of SOS (18). In addition, SOS^cat samples a broad range of turnover rates by fluctuating between distinct, long-lived functional states (19). Despite these advances, the nucleotide specificity of Ras bound to the active and allosteric sites of SOS and assembly with RAS, including oncogenic mutants, at the molecular level is poorly understood.Targeting oncogenic Ras mutants presents significant challenges because of their relatively smooth surface that lacks potentially druggable pockets (6). Nevertheless, the discovery of KRas inhibitors, particularly those that form irreversible covalent bonds with Cys-12, comprises one of the most active areas of cancer research (6, 20). As there are few windows of opportunity to specifically target Ras mutants (6), apart from covalent binding to Cys-12, other approaches have also been explored, such as designing molecules to disrupt Ras•SOS interactions, thereby preventing activation of Ras (20). An increasing number of small-molecule disruptors and peptide mimetics have been designed to disrupt the Ras•SOS interaction (21). Potent small-molecule disruptors have recently been discovered that inhibit the formation of the Ras•SOS complex and demonstrate antiproliferative activity, representing a viable approach for targeting Ras-driven tumors (22, 23). These results highlight the therapeutic importance of disrupting the interaction between Ras and SOS.Over the past three decades, native ion mobility–mass spectrometry (IM-MS) has evolved as a powerful analytical technique to investigate protein complexes and their interaction with other molecules (24–26). In native MS, biological samples in aqueous ammonium acetate are ionized using nanoelectrospray ionization and introduced into a mass spectrometer tuned to preserve noncovalent interactions and structure (24, 27). Native IM-MS can provide information on protein complexes, such as subunit stoichiometry and topology (28), and, unlike other biophysical techniques, resolve individual ligand-binding events (29). In combination with an apparatus to control temperature, native MS can be used to determine equilibrium binding constants and thermodynamics for protein–ligand and protein–protein that are in direct agreement with traditional biophysical approaches, such as isothermal calorimetry (30–35). Recently, native MS has been employed to determine transition state thermodynamics for the intrinsic GTPase activity of KRas and several oncogenic mutants (36). Notably, intrinsic GTPase activity rates determined using traditional solution-based assays mirrored those obtained using native MS (36).Although the interaction of Ras and SOS has been the subject of numerous studies, the interaction of oncogenic Ras mutants with SOS remains poorly described. To better understand the role of these interactions in cancer, native IM-MS is used to characterize the molecular assemblies formed between SOS^cat and mutants of KRas associated with cancer. IM spectrometry shows conformational heterogeneity of SOS^cat with specific conformers engaging KRas. Three selected oncogenic mutants of KRas form distinct molecular assemblies with SOS^cat, such as KRas^G13D forming exclusively a ternary complex with SOS^cat. The cryogenic electron microscopy (cryo-EM) structure of the KRas^G13D•SOS^cat complex provides insight into the mechanism for the higher affinity of KRas^G13D for SOS^cat. KRas^G13D–GTP also allosterically modulates the activity of SOS^cat more than the wild-type protein. In addition, the recent inhibitors developed to disrupt the KRas•SOS^cat complex, BAY-293 and BI-3406, cannot dissociate KRas^G13D•SOS^cat complexes. Other small molecules, such as ARS-1620 and Kobee0065, display a range of efficacies in disrupting complexes formed between KRas mutants and SOS^cat.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

Situated in a central position of the complex intracellular signaling network, Ras proteins play critical roles in regulating cell growth, differentiation, migration and apoptosis through cycling between the guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms (1, 2). Aberrant signaling caused by oncogenic mutations in Ras that break this physiological balance can result in uncontrolled cell proliferation and ultimately the development of human malignancies (3, 4). Despite its well-established role in tumorigenesis and the extensive efforts to target this oncoprotein in past decades, clinically approved therapies remain unavailable. One obstacle to the development of anti-Ras drugs lies in the native structures of active and inactive Ras that lack apparently druggable pockets for high-affinity interactions with inhibitory compounds (5–7).Both the active and inactive forms of Ras, however, are inherently flexible, populating rare conformers distinct from the native structures and presenting alternative opportunities for drug discovery (8–11). For example, in GTP-bound active Ras, a major and minor state (termed states 2 and 1, respectively) coexist in solution and exchange on a millisecond timescale, with state 1 showing surface roughness unobserved in the major state (12–18). The direct visibility of state 1 in the one-dimensional ³¹P NMR spectra of active Ras largely facilitated its early discovery and characterization (12, 19). And the available mutants of H-Ras (e.g., T35A), or the homolog M-Ras, which predominantly assume the state 1 conformation, further promoted the atomic-resolution studies of its structure and internal dynamics, as well as the concomitant drug discovery efforts targeting this low-populated conformer (17, 18, 20).In comparison to the intensive studies on active Ras, research on the dynamics of GDP-bound inactive Ras has lagged far behind, presumably due to its high degree of spectral homogeneity with little sign of resonance splitting or exchange broadening at room temperature (21). The previously reported cryptic pockets for covalent and noncovalent inhibitors of Ras•GDP (22–24), which are unseen in the compound-free structure, nevertheless indicate that the inactive form is also structurally plastic. The recent relaxation-based NMR experiments carried out at low temperature successfully captured the intrinsic microsecond timescale motions in Ras•GDP, which map to regions that overlap with those rearranged on the binding of inhibitors (11). However, the structural information of the transiently formed excited state, in the form of chemical shifts, is not available from the relaxation measurements, owing to the fast exchange rate on the chemical shift timescale. Moreover, unlike the case of active Ras, there are no known mutations that can stabilize the excited state of Ras•GDP for investigations using conventional biophysical techniques. Thus far, the sparsely populated conformations of inactive Ras derived from its microsecond dynamics remain poorly understood, precluding structure-based rational drug discovery.To address these challenges, in this work we constructed a solution ensemble of Ras•GDP by integrating atomistic computer simulation with diverse NMR experimental parameters containing complementary information about the intrinsic protein motions on timescales from picoseconds to microseconds. This NMR-based ensemble well covers the slow dynamics as probed by spin relaxation and provides an atomic-resolution delineation of thermally accessible conformations, including those bearing surface or buried pockets similar to the cryptic pockets previously observed in the inhibitor-bound forms. The utility of the Ras•GDP ensemble in the development of inhibitors is demonstrated by ensemble-based virtual screening, which achieves an impressive level of enrichment of known binders.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5) and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct-acting antivirals, but multiple public health and financial barriers (7, 8), along with the possibility of reinfection or continued disease progression (7, 9, 10), have resulted in a continued rise in HCV infections. An HCV vaccine remains essential to proactively protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (11, 12). The challenges posed by HCV sequence diversity (12, 13), glycan shielding (14, 15), immunodominant nonneutralizing epitopes (16–19), and preparation of a homogeneous E1E2 antigen all contribute to the difficulty in generating protective B cell immune responses. Although multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited (20). Optimization of E1E2 to improve its immunogenicity and elicitation of bnAbs through rational design may lead to an effective B cell-based vaccine (21).HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (22–24). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (25) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (24, 26, 27) and interactions between E1 and E2 ectodomains (28–30). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and SR-B1 coreceptors as part of a multistep entry process on the surface of hepatocytes (31–34). Neutralizing antibody responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (18, 35–40). Structural knowledge of bnAb antibody–antigen interactions, which often target E2 epitopes in distinct antigenic domains B, D, or E (18, 41, 42), can inform vaccine design efforts to induce bnAb responses against flexible HCV epitopes (43–45). E1E2 bnAbs, including AR4A, AR5A (46), and others recently identified (38), are not only among the most broadly neutralizing (35) but also represent E1E2 quaternary epitopes unique to antibody recognition of HCV.Although much is known about bnAb responses to E1E2 glycoproteins, induction of B cell-based immunity with an E1E2-based vaccine immunogen (47–49) has remained difficult. The inherent hydrophobicity of E1 and E2 TMDs (24, 50) may impede uniform production of an immunogenic E1E2 heterodimer that could be utilized for both vaccine development and E1E2 structural studies. Although partial E1 and E2 structures have been determined (39, 51–54), many other enveloped viruses have structures of a complete and near-native glycoprotein assembly (55–59), providing a basis for rational vaccine design (60–62). Viral glycoproteins of influenza hemagglutinin (63), respiratory syncytial virus (RSV) (55), severe acute respiratory syndrome coronavirus 2 (64), and others (65, 66) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. HIV gp120–gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (56, 67). Previously described E1E2 glycoprotein designs include covalently linked E1 and E2 ectodomains (68, 69), E1E2 with TMDs intact and an immunoglobulin G (IgG) Fc tag for purification (70), as well as E1 and E2 ectodomains with a cleavage site (68), which presented challenges for purification either due to intracellular expression or to high heterogeneity. Two recently described scaffolded E1E2 designs, while promising, have not been shown to engage monoclonal antibodies (mAbs) that recognize the native E1E2 assembly, though they were engaged by E1-specific and E2-specific mAbs, as well as coreceptors that recognize E2 (71). Therefore, these presentations of E1E2 glycoproteins may not represent a native and immunogenic heterodimeric assembly, and thus their potential as vaccine candidates remains unclear.Here, we describe the design of a secreted E1E2 glycoprotein (sE1E2) that mimics both the antigenicity in vitro and the immunogenicity in vivo of the native heterodimer through the scaffolding of E1E2 ectodomains. In testing our designs, we found that both replacing E1E2 TMDs with a leucine zipper scaffold and inserting a furin cleavage site between E1 and E2 enabled secretion and native-like sE1E2 assembly. We assessed the size, heterogeneity, antigenicity, and immunogenicity of this construct (identified as sE1E2.LZ) in comparison with full-length membrane-bound E1E2 (mbE1E2). sE1E2.LZ binds a broad panel of bnAbs to E2 and E1E2, as well as coreceptor CD81, providing evidence of assembly into a native-like heterodimer. An immunogenicity study indicated that sera of mice injected with sE1E2.LZ neutralize HCV pseudoparticles at levels comparable to sera from mice immunized with mbE1E2. This sE1E2 design is a form of the native E1E2 heterodimer that both improves upon current designs and represents a platform for structural characterization and engineering of additional HCV vaccine candidates.     

A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Intracellular chloride concentration ([Cl⁻]_i) is a major determinant of neuronal excitability, as synaptic inhibition is primarily mediated by chloride-permeable receptors (1). In the mature brain, [Cl⁻]_i is maintained at low levels by chloride extrusion, which renders γ-aminobutyric acid (GABA) hyperpolarizing (2) and counteracts activity-dependent chloride loads (3). GABAergic inhibition in the adult is crucial not only for preventing runaway excitation of glutamatergic cells (4) but also for entraining neuronal assemblies into oscillations underlying cognitive processing (5). However, the capacity of chloride extrusion is low during early brain development (6, 7). Additionally, immature neurons are equipped with chloride uptake mechanisms, particularly with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (8–12). NKCC1 contributes to the maintenance of high [Cl⁻]_i in the developing brain (13), favoring depolarization through GABA_A receptor (GABA_AR) activation in vivo (14, 15).When GABA acts as a depolarizing neurotransmitter, neural circuits generate burst-like spontaneous activity (16–20), which is crucial for their developmental refinement (21–24). In vitro evidence indicates that GABAergic interneurons promote neuronal synchrony in an NKCC1-dependent manner (10, 12, 25–28). However, the in vivo developmental functions of NKCC1 are far from understood (29, 30). One fundamental question is to what extent NKCC1 and GABAergic depolarization supports correlated spontaneous activity in the neonatal brain. In the neocortex, GABA imposes spatiotemporal inhibition on network activity already in the neonatal period (14, 25, 31, 32). Whether a similar situation applies to other brain regions is unknown, as two recent chemo- and optogenetic studies in the hippocampus yielded opposing results (25, 33). Manipulations of the chloride driving force are potentially suited to resolve these divergent findings, but pharmacological (34–36) or conventional knockout (10, 11, 37) strategies suffer from unspecific effects that complicate interpretations.Here, we overcome this limitation by selectively deleting Slc12a2 (encoding NKCC1) from telencephalic glutamatergic neurons. We show that chloride uptake via NKCC1 promotes synchronized activity in acute hippocampal slices, but has weak and event type-dependent effects in CA1 in vivo. Long-term loss of NKCC1 leads to subtle changes of network dynamics in the adult, leaving synaptic development unperturbed and behavioral performance intact. Our data suggest that NKCC1-dependent chloride uptake is largely dispensable for several key aspects of hippocampal development in vivo.     

DLL1 orchestrates CD8+ T cells to induce long-term vascular normalization and tumor regression

The immunosuppressive and hypoxic tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we showed that elevated levels of Delta-like 1 (DLL1) in the breast and lung TME induced long-term tumor vascular normalization to alleviate tumor hypoxia and promoted the accumulation of interferon γ (IFN-γ)–expressing CD8⁺ T cells and the polarization of M1-like macrophages. Moreover, increased DLL1 levels in the TME sensitized anti-cytotoxic T lymphocyte–associated protein 4 (anti-CTLA4) treatment in its resistant tumors, resulting in tumor regression and prolonged survival. Mechanically, in vivo depletion of CD8⁺ T cells or host IFN-γ deficiency reversed tumor growth inhibition and abrogated DLL1-induced tumor vascular normalization without affecting DLL1-mediated macrophage polarization. Together, these results demonstrate that elevated DLL1 levels in the TME promote durable tumor vascular normalization in a CD8⁺ T cell– and IFN-γ–dependent manner and potentiate anti-CTLA4 therapy. Our findings unveil DLL1 as a potential target to persistently normalize the TME to facilitate cancer immunotherapy.

One of the major challenges currently facing cancer treatments is the aberrant tumor microenvironment (TME), characterized as hypoxia, immunosuppression, acidity, and high interstitial fluid pressure (IFP) (1–5). These properties render tumors resistant to many kinds of cancer treatment modalities. High IFP prevents the penetration and distribution of drug agents into the tumor parenchyma, while hypoxia compromises the effectiveness of chemotherapy and radiotherapy because both treatment modalities often require reactive oxygen species to evoke antitumor activities (4, 6). In addition, hypoxia induces the secretion of multiple immune inhibitory factors and promotes the accumulation of immune regulatory cell populations, such as transforming growth factor-β (TGF-β), interleukin 10 (IL10), myeloid-derived suppressor cells (MDSCs), M2-like tumor-associated macrophages (M2-TAMs), and regulatory T cells (Tregs) (1, 3, 7–9). Thus, the hypoxic and immunosuppressive TME hinders cancer immunotherapy to efficiently eradicate cancer cells.Emerging evidence suggests that the abnormal tumor vasculature contributes largely to the aberrant TME (1, 3, 4). Tumor blood vessels are tortuous, dilated, and leaky with low pericyte coverage. The resulting blood flow is often static and fluctuated and therefore creates a hypoxic and acidic TME with high IFP (4). Therefore, tumor vascular normalization has been proposed as a promising approach to alleviate the aberrances within the TME, thus enhancing the efficacy of a range of cancer treatment modalities, including chemotherapy, radiotherapy, and immunotherapy (10–18). Vascular endothelial growth factor (VEGF) ligands and receptors constitute one of the most potent proangiogenic signaling pathways (19). Various VEGF signaling inhibitors, such as Bevacizumab and Cediranib, have been approved to treat several types of cancers. VEGF signaling inhibitors can induce tumor vascular normalization; however, the duration of the normalization is usually transient, and therefore, the improvement to the concurrent chemotherapy and immunotherapy is marginal (4, 19–21). In addition, many kinds of cancer are intrinsically resistant to VEGF signaling targeted therapy (4, 19). Thus, novel approaches are needed to induce tumor vascular normalization for longer periods and in broad tumor types.The evolutionarily conserved Notch signaling pathway plays critical roles in cell differentiation and blood vessel formation. The Notch signaling pathway consists of four Notch receptors (Notch 1 to 4) and four ligands (Jagged1, Jagged2, Delta-like 1 [DLL1], and DLL4) in murine (22). Both Notch receptors and ligands are membrane proteins. DLL1, DLL4, and Jagged1 have been shown to express in endothelial cells and play important roles in vascular development and postnatal vessel formation (23, 24). DLL1 and DLL4 are also associated with tumor angiogenesis (24–26). DLL4 is usually expressed in tumor endothelial cells but rarely in tumor cells (27, 28). Blockade of DLL4 suppresses tumor growth through the induction of nonfunctional tumor vessel formation (24, 25, 29). Thus, activation of DLL4/Notch signaling has the potential to increase tumor vascular maturation. Indeed, higher expression of DLL4 in bladder tumor endothelial cells was correlated with vessel maturation (30). Unfortunately, long-term DLL4 blockade led to vascular neoplasms, and persistent activation of DLL4/Notch signaling promoted T cell acute lymphoblastic leukemia (T-ALL) (31–33).Because of these potential safety concerns of chronic blockade or activation of DLL4/Notch signaling, we proposed instead to remodel tumor vessels via the activation of DLL1/Notch signaling. In contrast to the extensive attention of DLL4 in tumor angiogenesis, the roles of DLL1 in tumor vessel formation is largely unknown. Here, we showed that overexpression of DLL1 in EO771 breast and LAP0297 lung tumor cells not only induced durable tumor vascular normalization but also stimulated CD8⁺ T cell activities. Interestingly, in vivo depletion of CD8⁺ T cells prior to tumor implantation or host IFN-γ deficiency abrogated the effects of DLL1 overexpression on tumor vessels, suggesting that selective activation of DLL1/Notch signaling induces long-term tumor vascular normalization via T cell activation. Moreover, DLL1/Notch signaling activation in combination with anti-CTLA4 therapy prolonged survival. Thus, this study uncovered DLL1 as a potential target to induce long-term tumor vascular normalization to enhance cancer immunotherapy.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (T_m of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (T_m of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the luminal surface of capillaries plays an important role in the delivery of lipid nutrients to vital tissues (e.g., heart, skeletal muscle, adipose tissue). A complex of lipoprotein lipase (LPL) and its endothelial cell transporter, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), is responsible for the margination of TRLs and their lipolytic processing (1–3). The importance of the LPL•GPIHBP1 complex for TRL processing is underscored by the development of severe hypertriglyceridemia (chylomicronemia) with loss-of-function mutations in LPL or GPIHBP1 or with GPIHBP1 autoantibodies that disrupt GPIHBP1•LPL interactions (4–7). Chylomicronemia is associated with a high risk for acute pancreatitis, which is debilitating and often life threatening (8, 9). Interestingly, increased efficiency of plasma triglyceride processing appears to be beneficial, reducing both plasma triglyceride levels and the risk for coronary heart disease (CHD). For example, genome-wide population studies have revealed that single-nucleotide polymorphisms that limit the ability of angiopoietin-like proteins 3 or 4 (ANGPTLs) to inhibit LPL are associated with lower plasma triglyceride levels and a reduced risk of CHD (10–14).GPIHBP1 is an atypical member of the LU domain superfamily because it contains a long intrinsically disordered and highly acidic N-terminal extension in addition to a canonical disulfide-rich three-fingered LU domain (15). At the abluminal surface of capillaries, GPIHBP1 is responsible for capturing LPL from heparan sulfate proteoglycans (HSPGs) in the subendothelial spaces and shuttling it to its site of action in the capillary lumen (3, 16). The capture of LPL from subendothelial HSPGs depends on electrostatic interactions with GPIHBP1’s intrinsically disordered acidic domain and stable hydrophobic interactions with GPIHBP1’s LU domain (15, 17, 18). In the setting of GPIHBP1 deficiency, LPL never reaches the capillary lumen and remains mislocalized, bound to HSPGs, in the subendothelial spaces. Aside from promoting the formation of GPIHBP1•LPL complexes, the acidic domain plays an important role in preserving LPL activity. The acidic domain is positioned to form a fuzzy complex with a large basic patch on the surface of LPL, which is formed by the confluence of several heparin-binding motifs. This electrostatic interaction stabilizes LPL structure and activity, even in the face of physiologic inhibitors of LPL (e.g., ANGPTL4) (19–22).Distinct expression profiles for ANGPTL-3, -4, and -8 underlie the tissue-specific regulation of LPL and serve to match the supply of lipoprotein-derived lipid nutrients to the metabolic demands of nearby tissues (21, 23–29). In the fasted state, ANGPTL4 inhibits LPL activity in adipose tissue, resulting in increased delivery of lipid nutrients to oxidative tissues. In the fed state, ANGPTL3•ANGPTL8 complexes inhibit LPL activity in oxidative tissues and thereby channel lipid delivery to adipocytes. While the physiologic relevance of tissue-specific LPL regulation is clear, the mechanisms by which ANGPTL proteins inhibit LPL activity remain both incompletely understood and controversial. One view holds that ANGPTL4 inhibits LPL activity by a reversible mechanism (30, 31). An opposing view, formulated early on by the laboratory of Gunilla Olivecrona, is that ANGPTL4 irreversibly inhibits LPL by a “molecular unfolding chaperone-like mechanism” (32). Hydrogen–deuterium exchange mass spectrometry (HDX-MS) studies have supported the latter view. Recent studies by our group revealed that ANGPTL4 catalyzes the irreversible unfolding (and inactivation) of LPL’s α/β-hydrolase domain and that the unfolding is substantially mitigated by the binding of GPIHBP1 to LPL (17, 19, 22). We further showed that ANGPTL4 functions by unfolding catalytically active LPL monomers rather than by promoting the dissociation of catalytically active LPL homodimers (33, 34). Despite these newer findings, a host of issues remains unresolved. For example, the binding site for ANGPTL4 on LPL has been controversial (31, 35); the initial conformational changes induced by ANGPTL4 binding have not been delineated, and how the early conformational changes in LPL progress to irreversible inactivation is unknown. In the current studies, we show, using time-resolved HDX-MS, that ANGPTL4 binds to LPL sequences proximal to the entrance of LPL’s catalytic pocket. That binding event triggers alterations in the dynamics of LPL secondary structure elements that are central to the architecture of the catalytic triad. Progression of those conformational changes leads to irreversible unfolding and collapse of LPL’s catalytic pocket. The binding of GPIHBP1 to LPL limits the progression of these allosteric changes, explaining why GPIHBP1 protects LPL from ANGPTL4-induced inhibition.     

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell–mediated destruction of insulin-producing β-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Type 1 diabetes (T1D) is a prototypical organ-specific autoimmune disease that develops in stages (1, 2). The stages are marked by the presence of islet autoantibodies directed against insulin and other β-cell proteins, followed by impaired glucose tolerance, and finally clinical diabetes marked by hyperglycemia and the need for insulin treatment (3). The T1D disease course provides a defined preclinical period and the ability to measure immune responses prior to clinical symptoms.Self-reactive T cells target pancreatic β-cells in both murine models of spontaneous autoimmune diabetes and human T1D (4), with a number of antigens implicated as T-cell epitopes (5, 6). Recently, posttranslationally modified (PTM) epitopes have been characterized as novel autoantigens that may lead to a break in tolerance, thus resulting in T-cell–mediated immunity to pancreatic islets. PTM of antigens is well-described in autoimmune diseases, such as celiac disease (gluten sensitivity), in which tissue transglutaminase mediates deamidation of glutamine to glutamic acid within gliadin to create immunogenic CD4 T-cell epitopes (7–10). In rheumatoid arthritis, citrullinated peptides form epitopes from cartilage proteins that both elicit antibody responses and activate CD4 T cells (11). Similarly, a novel class of epitopes within T1D are hybrid insulin peptides (HIPs) that are formed within lysozymes of β-cells through a covalent bond between an insulin peptide fragment and another β-cell peptide, thereby generating a neo-epitope (12, 13).Recent studies provide strong evidence for the role of HIPs in the development of diabetes in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes (12, 14–16). Notably, the antigen for the well-studied “diabetogenic” BDC2.5 T-cell clone and transgenic mouse model is a HIP formed between a peptide fusion of C-peptide and a cleavage product of chromogranin A, termed WE14 (12, 17, 18). C-peptide is cleaved from the A and B chains of insulin prior to secretion from the β-cell. In the NOD mouse, HIP-reactive CD4 T cells have a proinflammatory phenotype, can be detected prior to the onset of diabetes, and their frequency increases as the disease progresses (15). Another CD4 T-cell epitope critical for NOD diabetes development is a fragment of the insulin B chain, consisting of amino acids 9 to 23 (B:9–23) (19–21). A strongly stimulating T-cell epitope is very likely a HIP consisting of a fragment of this insulin B-chain peptide with a portion of C-peptide fused to the C-terminal end (22). HIP-reactive CD4 T cells have also been studied in the context of human T1D, with multiple CD4 T-cell clones and lines grown from the residual pancreatic islets of T1D organ donors subsequently responding to these neo-epitopes (12, 23, 24). A number of HIP-reactive T cells have also been measured from the peripheral blood in newly diagnosed T1D patients (25–30); however, the timing of when these T cells appear in the disease course and whether these responses directed at PTM peptides precede those toward native antigens remains to be addressed. We hypothesized that HIP T-cell responses precede clinical diabetes development and are more robust than responses to native insulin peptides.In this study, we longitudinally collected peripheral blood mononuclear cells (PBMCs) from genetically at-risk individuals and measured reactivity to a panel of HIPs and native antigens using sensitive enzyme-linked immunospot (ELISPOT) assays, which have previously been used to identify CD4 T-cell responses in T1D (31). We show that PBMCs respond to native insulin peptides, but the cells respond more robustly to specific HIPs, including the insulin B chain B:9–23 HIP (B22E) and two C-peptide–derived HIPs (C-peptide/islet amyloid polypeptide-2 [C:IAPP-2] and C:A chain). We demonstrate that T-cell responses fluctuate between pro- and antiinflammatory during the preclinical phase prior to T1D development. Interestingly, individuals who progressed to clinical disease or who seroconverted to islet autoantibody positivity during the course of the study had a distinct polarization toward proinflammatory responses to specific HIPs. Remarkably, the T-cell response to the C:IAPP-2 HIP correlated with worsening measures of blood glucose. Overall, the data support a pathogenic role for PTM epitopes in the preclinical stage of T1D, and the fluctuating nature of the T-cell responses has implications for timing therapies to prevent T1D and potentially other autoimmune disorders.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate–BCoA–CPT1A axis for the regulation of immune homeostasis.

Regulatory T (Treg) cells are CD4⁺ T cells expressing Foxp3 that play a key role in immune suppression (1–3). They can be divided into natural Treg (nTreg) and inducible Treg (iTreg) cells (1–3). nTreg cells, which are often referred to as thymic Treg (tTreg), arise during CD4⁺ T cell differentiation in the thymus under the influence of relatively high-avidity interactions of the T cell receptor (TCR) with self-antigens (1–3). iTreg cells, also called peripherally induced Treg (pTreg), develop in secondary lymphoid tissues. In the presence of TGFβ1, naive CD4⁺ T are induced into iTreg cells upon TCR ligation and costimulation by antigen-presenting cells (APCs) in response to non-self antigens, such as allergens, food, and the commensal microbiota (1–3).It has been demonstrated that iTreg cells are enriched in gut-associated lymphoid tissues (GALTs) and are important for the maintenance of intestinal immune homeostasis (3–5). Intestinal iTreg cells were found to be important for the regulation of inflammatory bowel diseases (IBDs), such as Crohn’s disease (CD) and ulcerative colitis (UC), which can potentially affect any portion of the gastrointestinal tract and induce many further complications such as tissue fibrosis, stenosis, fistulas, and colon cancer over time (6). Enhancement of intestinal iTreg function or adoptive transfer of iTreg could significantly alleviate IBDs in mice (7–9).Different types of T cells are featured by distinct metabolic characteristics. Unlike effector CD4⁺ T cells (Teffs), including Th1, Th2, Th9, and Th17 cells, that are mainly reliant on aerobic glycolysis, iTreg cells largely rely on fatty acid oxidation (FAO) (10–12). Accumulating evidence has demonstrated that T cell differentiation is always coupled with metabolic reprogramming (13, 14). For instance, FAO needs to be established in the process of iTreg differentiation. Up-regulation of FAO improved iTreg generation, whereas impairment in FAO compromised iTreg differentiation (12, 13, 15, 16).FAO, comprised of a cyclical series of reactions, demands different fatty acids (FAs), which can be divided into long-, medium-, and short-chain fatty acids (LCFAs, SCFAs, and MCFAs). It dominantly occurs in mitochondria and results in acetyl-CoA (AcCoA), which could be consumed in tricarboxylic acid (TCA) cycle. For the oxidation of LCFA, it initiates from LCFA activation in cytoplasm, resulting in long-chain acyl-CoA. Subsequently, these resulting molecules are converted into long-chain acyl-carnitine by carnitine palmitoyltransferase 1 (CPT1), which is anchored on the mitochondrial outer membrane. Following its shuttling into mitochondria, long-chain acyl-carnitine experiences a chain of reactions to support FAO. Apparently, the transportation of LCFA from cytoplasm into mitochondria is a prerequisite for FAO. CPT1, the rate-limiting enzyme controlling this key step, is thus recognized as a determinant for FAO. In contrast, SCFAs and MCFAs can diffuse across mitochondrial membrane and drive FAO in a CPT1-independent manner (17). Nevertheless, extensive investigations have suggested an important role for CPT1 in iTreg differentiation (12, 13, 15, 16).In recent years, butyrate, a specific type of SCFA produced from fiber-rich diets through microbial fermentation, was shown to play a critical role in the maintenance of intestinal homeostasis and was therefore recognized as an effective ingredient from food (18–24). By modulating distinct types of immune cells, including dendritic cells (18, 19), macrophages (20), and B and T cells (21–24), butyrate contributes to the orchestration of the delicate balance in intestinal immune system. Elegant investigations have elucidated that butyrate is able to facilitate iTreg differentiation by up-regulating Foxp3 expression as a histone deacetylase (HDAC) inhibitor (22, 23). Meanwhile, butyrate, as metabolic fuel and energy source, could also support FAO in colonic epithelial cells (25). However, whether butyrate could regulate FAO to promote iTreg differentiation is unclear.In this study, we found that increased FAO contributed to enhanced iTreg cell differentiation in response to butyrate. Butyrate was processed, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which played a critical role in the control of FAO by targeting CPT1A. We found that BCoA competed with malonyl-CoA (MCoA), the best-known metabolic intermediate inhibiting CPT1A, to unleash CPT1A activity for FAO and thereby iTreg differentiation. Inhibition of ACSS2 to block BCoA generation compromised butyrate-mediated iTreg generation as well as mitigation of mouse colitis. Collectively, we depicted a previously unappreciated mechanism, namely, the butyrate–BCoA–CPT1A regulatory axis, for iTreg differentiation.     

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against β-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.

Two of the most widely studied proteins that regulate T cell activation are cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1). Inhibiting them can unleash regulatory controls of T cell activation, allowing the T cells to display their full functional potential. CTLA-4 and PD-1 are extensively studied in the cancer field, where their inhibitors—monoclonal antibodies—are used to treat cancer patients. Despite achieving success clinically, the checkpoint blockade immunotherapy can result in immune-related adverse events that frequently include endocrine autoimmune diseases (1), among them type 1 diabetes (T1D) (2–4). A recent comprehensive review summarized the clinical features of T1D induced by the immune checkpoint inhibition (4).To better understand how PD-1 is regulating T1D, we examine here the nonobese diabetic (NOD) mouse model for changes in various cellular components following PD-1 blockade. PD-1 is synthesized de novo in activated T cells mediated by T cell receptor (TCR) signaling (5–7). Whereas PD-1 expression is rapidly up-regulated after antigen stimulation of naïve T cells, sustained TCR stimulation results in substantially higher expression of PD-1 and the establishment of T cell exhaustion in the examples of chronic viral infection and cancers (8–13). NOD mice with a gene knockout of PD-1 or treated with PD-1 blocking antibody develop accelerated autoimmune diabetes shown in a number of studies (14–21). Both autoreactive CD4 and CD8 T cells can respond to PD-1/PD-L1 blockade and contribute to the acute diabetes development (17–19). Central in this process is PD-L1 (the ligand of PD-1) expressed by the islet parenchymal cells (20, 21), which limits the T cell function in the islets and protects against autoimmune diabetes.In this report we examine changes in various cellular components following PD-1 blockade by single-cell RNA sequencing (scRNA-seq) and identify a previously unexplored islet macrophage population derived from monocytes with high proinflammatory activity. In the islets of anti–PD-1–treated mice, the infiltration by monocyte-derived macrophage (MoMac) was under the influence of CD4 and particularly CD8 T cells. The CD8 T cells largely comprised the precursor exhausted T (T_PEX) cells that were activated and differentiated to produce abundant IFN-γ in response to PD-1 blockade, which in turn activated the infiltrated MoMac to promote diabetes progression. The anti–PD-1 induced development of acute diabetes was reduced by restricting the infiltration and function of such MoMac in islets. Our study establishes that the myeloid cell compartment is an indispensable component of PD-1 regulation in autoimmune diabetes. Our study provides a cellular target, the MoMac, that may minimize the adverse effects of checkpoint blockade immunotherapy.