Crucial role of nonspecific interactions in amyloid nucleation

Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.During the process of amyloid formation, normally soluble proteins assemble into fibrils that are enriched in β-sheet content and have diameters of a few nanometers and lengths up to several micrometers. This phenomenon has been implicated in a variety of pathogenic processes, including Alzheimer’s and Parkinson’s diseases, type 2 diabetes, and systemic amyloidoses (1–3). The association with human diseases has largely motivated a long-standing effort to probe the assembly process, and numerous studies have aimed at elucidating the mechanism of amyloid aggregation (4). The basic nature of the aggregation reaction has emerged as a nucleation and growth process (5, 6), where the aggregates are created through a not well-understood primary nucleation event and can grow by recruiting additional peptides or proteins to their ends (7, 8). In this paper, we focus on the nature of this primary step in amyloid nucleation and the fundamental initial events that underlie amyloid formation.Amyloidogenic peptides and proteins, when in their nonpathological cellular form, can range in the structures from mainly α-helical to β-sheet and even random coil, whereas the amyloid forms of proteins possess a generic cross–β-structure (9–14). The formation of amyloid is, hence, accompanied by marked changes in the conformations of the peptides and proteins that undergo this process. A pertinent question is whether this conformational change takes place simultaneously with the nucleation process or whether nucleation takes place first and is then followed by conformational change. These two possible scenarios of nucleation have been extensively discussed in the experimental and theoretical literature (5, 8, 15–19). We will refer in this work to the two scenarios simply as one-step nucleation (1SN), in which the β-sheet–enriched nucleus forms directly from the solution, and two-step nucleation (2SN), where soluble monomers first assemble into disordered oligomers, which subsequently convert into a β-sheet nucleus. Disordered oligomers, ranging in size between dimers and micrometer-sized particles, have been observed in some experiments (20–28). These findings highlight a central question regarding the role of disordered oligomers in fibril formation: are such clusters a necessary step in the process of fibril formation or just a byproduct?From a biological and biomedical perspective, it is important to understand the conditions under which oligomeric clusters form, because such species exhibit high cytotoxicity (1, 29–31). Indeed, there is strong evidence that the disordered oligomers rather than fully grown fibrils are the main pathogenic species in protein aggregation diseases (31–33). As such, defining the role of the prefibrillar oligomers during amyloid formation will be crucial to develop intervention strategies that target these species (1, 30, 34, 35).Mutations in the polypeptide sequence and extrinsic changes in the experimental conditions are known to alter the concentrations of aggregated species, their size, and their cytotoxicity (25, 36–39). For instance, mutations that increase hydrophobicity of the Alzheimer’s β-peptide (Aβ1–42) have a pronounced effect on its aggregation behavior and the size distribution of the resulting oligomers (23–26, 40), promoting toxicity and expediting the fibrillization process. In the same spirit, two extra hydrophobic residues in Aβ1–42 are believed to contribute to the more pronounced oligomerization and faster fibrillization compared with its alloform Aβ1–40 (24, 25, 40). Temperature, pH, and concentration of certain metals also affect oligomerization and pathways of fibrillization (41–44).The common feature of the above experiments is that they modify the internal free energy difference between the soluble and the β-sheet–forming state, also called the β-sheet propensity, which has been extensively studied in the literature (45–48). However, they also modify interactions between peptides that aggregate, a crucial contribution that has not yet been systematically addressed.In this paper, we study the effect of nonspecific interactions between peptides on the amyloid nucleation process. Such nonspecific interactions do not depend on the atomistic details of the amino acids involved, allowing us to address question about amyloid aggregation and nucleation using a coarse-grained model. In particular, generic hydrophobic stretches in the sequence of Aβ have been shown to be sufficient to promote aggregation (49, 50). Mutations of nonpolar residues to other nonpolar residues had little or no effect on aggregation, whereas mutations that reduce charge and/or increase hydrophobicity enhanced it (50, 51). Furthermore, atomic force microscopy measurements have shown that the strength of overall interactions between amyloidogenic proteins correlates with their tendency to aggregate (52, 53).We have performed extensive computer simulations that allowed us to observe both the 1SN and the 2SN mechanisms. These simulations reveal that 1SN and 2SN can be viewed as two limits of the same process, something that several previous studies have suspected (16, 18). Importantly, we observe that only 2SN is possible at low peptide concentrations, comparable with the levels that are found in vivo. Another key observation is that fibril nucleation typically does not proceed through the most prevalent oligomeric species but rather, through an oligomer with a size that is only observed as a result of rare fluctuations. As a consequence, such oligomers will be hard to capture experimentally, although their presence is required for nucleation to take place. Our simulations show that the free energy barrier for fibril nucleation through the two-step mechanism decreases with increasing strength of the interpeptide interactions. Furthermore, the critical nucleus size in the two-step mechanism is found to grow with the increase in the peptide concentrations as well as with stronger interpeptide interactions, which is in direct contrast with the classical nucleation. These results imply that weakening the nonspecific interactions between peptide monomers in solution and thereby, simultaneously increasing both the free energy barrier for oligomer formation and the free energy barrier for peptide conversion at a given oligomer size may be a crucial step in preventing amyloid aggregation.     

Identification of divergent type VI secretion effectors using a conserved chaperone domain

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.Protein secretion systems play a pivotal role in bacterial interspecies interaction and virulence (1, 2). Of the known secretion systems in Gram-negative bacteria, the type VI secretion system (T6SS) enables bacteria to compete with both eukaryotic and prokaryotic species through delivery of toxic effectors (2–4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, including many important pathogens (2, 8), and has been implicated as a critical factor in niche competition (9–11).The T6SS structure is composed of an Hcp inner tube, a VipAB outer sheath that wraps around the Hcp tube, a tip complex consisting of VgrG and PAAR proteins, and a membrane-bound baseplate (2, 4, 12). Sheath contraction drives the inner Hcp tube and the tip proteins, VgrG and PAAR, outward into the environment and neighboring cells (13, 14). The contracted sheath is then dissembled by an ATPase ClpV and recycled for another T6SS assembly and contraction event (12, 15, 16). Two essential T6SS baseplate components, VasF and VasK, are homologous to the DotU and IcmF proteins of the type IV secretion system (T4SS) in Legionella pneumophila (17).Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and PAAR genes abolishes T6SS secretion (14). Some VgrG and PAAR proteins carry functional extension domains and thus act as secreted T6SS effectors, as exemplified by the VgrG1 actin cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae (18, 19), and the nuclease domain of the PAAR protein RhsA in Dickeya dadantii (20). Known T6SS effectors can target a number of essential cellular components, including the actin and membrane of eukaryotic cells (18, 21, 22) and the cell wall, membrane, and DNA of bacterial cells (3, 18–20, 23, 24). Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26, 27).For non-VgrG/PAAR–related effectors, their translocation requires either binding to the inner tube Hcp proteins as chaperones or binding to the tip VgrG proteins (2, 14, 28). T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3, 29–31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging. A heuristic approach based on the physical properties of effectors has been used to identify a superfamily of peptidoglycan-degrading effectors in bacteria (32). A recent study identified a common N-terminal motif in a number of T6SS effectors (31). However, this motif does not exist in the T6SS effector TseL in V. cholerae (18).In this study, we report that VC1417, the gene upstream of tseL, encodes a protein with a highly conserved domain, DUF4123. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain proteins are generally located upstream of genes encoding putative T6SS effectors. Using the conserved domain sequence, we bioinformatically predicted a large family of effector proteins with diverse functions in Gram-negative bacteria. We validated our prediction by the identification and characterization of a new secreted effector TseC and its antagonistic immunity protein TsiC in Aeromonas hydrophila SSU. Our results demonstrate a new effective approach to identify T6SS effectors with highly divergent sequences.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.Due to their sessile lifestyle, the development of plants is characterized by continuous growth, generating the capacity to adapt to environmental conditions. Such flexibility has been made possible by a set of morphological adjustments that are accomplished through altered growth regulation of different plant organs, such as leaves or roots. Most aspects of plant development are regulated by the differential distribution of the plant hormone indole-3-acetic acid (IAA, or auxin) between cells or tissues (reviewed by ref. 1). The formation of auxin maxima is generated concomitantly by local auxin biosynthesis, metabolism, and directional transport (2–8).Polar auxin transport occurs in a cell-to-cell manner and is dependent on plasma membrane-localized auxin influx and efflux carriers (reviewed by ref. 9). Among them, the PIN-FORMED (PIN) auxin efflux carriers are essential for plant development, and single or multiple pin mutants display phenotypes typical for auxin transport defects, such as tropism, embryo development, organogenesis, and root meristem patterning defects (6, 7, 10–14). A polar subcellular localization has been shown for most of the plasma membrane-localized auxin transporters, in particular for the PIN proteins (PIN1-4 and PIN7) and, to some extent, also for the ATP-BINDING CASSETTE SUBFAMILY B proteins (ABCBs) and AUXIN RESISTANT 1 (AUX1) (11–13, 15–20). The PIN proteins are known to be essential for targeting and redirecting auxin flux, which modulates the spatial pattern of expression of auxin response markers (21). PINs can be targeted toward the apical (shootward), basal (rootward), or lateral plasma membrane depending upon the PIN protein identity, the cell type, and the developmental context (reviewed by ref. 22). In the root, PIN1 is localized basally toward the root tip in stele provascular cells (12). PIN2 is also localized basally in young cortex cells close to the root meristem but is localized apically in mature cortex cells, epidermal cells, and the lateral root cap (16, 22, 23).Until now, it has been unclear whether newly synthesized PIN proteins are initially secreted to the plasma membrane in a polar or apolar manner. In Arabidopsis thaliana, the current model for PIN polar localization establishment and maintenance at the plasma membrane is based on endocytosis, polar recycling, and restriction of lateral diffusion (reviewed by ref. 24). PIN proteins are internalized via clathrin-mediated endocytosis (25, 26) and can cycle back to plasma membrane domains via distinct trafficking routes. Recycling and endocytosis of PIN1 depend on the endosome-localized fraction of the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM (27, 28), which is sensitive to the fungal toxin brefeldin A (BFA) (29). ARF-GEFs are essential regulators of vesicle formation and, among the eight ARF-GEFs in Arabidopsis, GNOM is the only one reported as being essential specifically for basal PIN recycling, whereas apical PIN and AUX1 localization and dynamics are not affected in gnom mutants (30, 31). Additionally, although apical targeting of AUX1 is resistant to BFA, subcellular AUX1 trafficking is BFA-sensitive, suggesting that trafficking of apical proteins may require both BFA-sensitive and -insensitive, GNOM-independent, ARF-GEF–mediated pathways (30, 32).In addition to GNOM, other Arabidopsis ARF-GEFs have been characterized, including GNOM-LIKE 1 (GNL1), which localizes to Golgi stacks and is BFA-resistant (33, 34). GNL1 acts in the early secretory pathway where it regulates COPI-mediated recycling of endoplasmic reticulum (ER)–resident proteins from the Golgi back to the ER (33, 34). Moreover, GNOM has recently been shown to predominantly localize to Golgi stacks (35) where it plays a minor but redundant function to GNL1 in ER-Golgi trafficking (33). The other Arabidopsis ARF-GEFs include GNL2, which is expressed specifically in pollen (36), and the five BIG ARF-GEFs, BIG1 to -5. BIG5, which is BFA-sensitive, has been described under the name BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE 1 (BEN1) as mediating early endosomal trafficking (37). BIG1 to -4, of which BIG3 is BFA-resistant whereas BIG1, -2, and -4 are BFA-sensitive, have recently been described as acting redundantly in the late secretory pathway from the trans Golgi network (TGN) to the plasma membrane, as well as in late vacuolar trafficking (38).Endosomal PIN homeostasis is tightly controlled by the retromer complex through the regulation of PIN protein trafficking to the vacuole, thus controlling polar PIN abundance within the cell (39–43). Additionally, a large amount of data has demonstrated that not only trafficking routes per se are essential to determine the polar localization of PIN proteins but also internal protein signals such as posttranslational phosphorylation via the protein kinase PINOID (PID) and the protein phosphatase 2A (PP2A) (44–46). Despite recent progress, our understanding of the mechanisms establishing basal polarity remains limited. In the present work, we aimed to unravel the details of PIN basal polarity establishment by identifying selective inhibitors of this process.A number of genetic screens have been successfully used to discover new components of the endomembrane system (for examples, see refs. 34, 37, and 47–51). However, most of the molecular actors regulating endomembrane trafficking are either essential to plant survival or belong to large protein families, leading to lethality of knock-out mutants or lack of a phenotype due to redundancy. The use of fast-acting molecules suitable for the highly dynamic nature of the endomembrane system circumvents these problems and has deepened our understanding of interconnected networks of trafficking routes (52–58). While BFA has expanded our knowledge of the GNOM-dependent recycling pathway (27), other small compounds can be used to dissect different trafficking routes. In recent studies, automated screening of small molecules based on inhibition of tobacco pollen tube growth led to the isolation of a set of compounds interfering with the endomembrane system (52). Through the screening of 46,418 diverse molecules, 360 were identified as inhibitors of pollen germination (53). To dissect the trafficking routes of plasma membrane proteins specifically, a secondary screen was established based on confocal laser-scanning microscopy, leading to the identification of 123 compounds named plasma membrane recycling compound set A (PMRA), which induce mislocalization of plasma membrane markers in the Arabidopsis root meristem (53).In the present study, we reasoned that using the PMRA endomembrane trafficking modulators in combination with BFA could unravel trafficking routes regulating basal plasma membrane targeting. We designed a chemical screen to identify PMRA molecules that modulated the accumulation of PIN1 in BFA-induced agglomerations. We subsequently identified the endosidin 8 (ES8) compounds, including the original compound ES8.0 and its more potent analog ES8.1, which selectively modify PIN1 basal plasma membrane targeting in Arabidopsis with minimal effects on apical plasma membrane proteins. Using this pharmacological approach, we herein confirm that GNOM plays a role in ER-Golgi trafficking independently of its role in recycling and reveal that a GNL1/GNOM-dependent early secretory pathway is essential for targeting PIN1 toward the basal plasma membrane. Furthermore, we demonstrate that this pathway is specific for basal polarity establishment, revealing an essential and previously unknown regulatory mechanism for establishing cell polarity and regulating auxin transport and plant development.