Molecular basis for plasma membrane recruitment of PI4KA by EFR3.

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIα/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease. One sentence summary: The cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA complex reveals the molecular mechanism underlying PI4KA membrane localisation, and provides novel insight into PI4KA regulation and its involvement in human disease.

Examining the longitudinal associations between activity limitations, instrumental supports and social participation in osteoarthritis: A CLSA population-based study.

OBJECTIVE: In osteoarthritis (OA) research, disability is largely studied within the context of activities of daily living. Broader consequences for social participation are often overlooked. In prior work, instrumental supports received and their perceived availability were shown to play a role in the maintenance of social participation. Two indicators of social participation were identified, diversity and intensity. The current study extends the findings from this prior cross-sectional work by examining these relationships longitudinally. METHODS: Data are from the baseline and 3-year follow-up questionnaires of the Canadian Longitudinal Study on Aging, a population-based study of people ages 45-85 years at baseline. The sample was restricted to those who at baseline reported a doctor diagnosis of OA (n = 4104). Using structural equation modeling, latent variables were derived at each time point for activity limitations, instrumental supports perceived and received, and social participation diversity and intensity. Longitudinal factorial invariance was assessed. Model covariates included age, sex, education, income, marital status, smoking status, obesity, and number of chronic conditions. RESULTS: For all latent variables, strong factorial longitudinal invariance was found. Activity limitations increased over time. Greater baseline social participation intensity was associated with increases in later intensity and diversity. Increasing activity limitations were associated with decreases in social participation and with increasing receipt of instrumental supports; they were not associated with changes in perceived availability of supports. However, increasing perceived availability was positively associated with social participation intensity. CONCLUSIONS: With a goal of increasing social participation, findings suggest a focus on interventions to reduce activity limitations in OA is necessary. Findings additionally highlight an important role for perceived availability of instrumental supports in maintaining or improving social participation in OA, in addition to current social participation, particularly intensity, for future social participation status.

Molecular structures and function of the autophagosome-lysosome fusion machinery.

Macroautophagy (also known as autophagy) plays a pivotal role in maintaining cellular homeostasis. The terminal step of the multi-step autophagy degradation pathway involves fusion between the cargo-laden, double-membraned autophagosome and the lytic organelle lysosome/vacuole. Over the past decade, various core components of the molecular machinery that execute this critical terminal autophagy event have been identified. This review highlights recent advances in understanding the molecular structures, biochemical functions, and regulatory mechanisms of key components of this highly sophisticated machinery including the SNARE fusogens, tethering factors, Rab GTPases and associated guanine nucleotide exchange factors, and other accessory factors.

Clinical effectiveness of mindfulness-based music therapy on improving emotional regulation in blind older women: A randomized controlled trial.

Background: This study aimed to investigate clinical effectiveness of a structured eight-week mindfulness-based music therapy (MBMT) program on improving mood regulation in older women with blindness. This investigation compared a MBMT group with a mindfulness intervention (MI) group and a control group. Methods: Ninety-two older females with blindness from a residential setting in Hong Kong were recruited and randomly allocated to a MBMT (n = 31), MI (n = 30), or control (n = 31) group. Psychological measurements regarding mood regulation and general mood states (namely, Difficulties in Emotion Regulation Scale [DERS], Geriatric Depression Scale [GDS], and Depression Anxiety Stress Scales-21), were taken at pretest and posttest. Outcome assessors were blinded to group assignment. Results: Data was analyzed based on intention-to-treat basis. At posttest, DERS scores in the MBMT group (mean differences and 95% confidence interval: 12.1, 5.5 to 18.8) and the MI group (7.2, 0.5 to 13.8) were lower than that in the control group. GDS scores in the MBMT group (2.9, 1.7 to 4.0) and the MI group (1.7, 0.6 to 2.9) were lower than those in the control group. Compared with the MI group, the MBMT group improved emotional awareness sub-scores in DERS (2.1, 0.2 to 4.1) and appeared to lower depression in GDS scores (1.1, -0.0 to 2.3; p = 0.053). Conclusion: MBMT seems more beneficial than MI alone for improving emotional regulation in older women with blindness. The combination of mindfulness and music can generate a synergetic effect by enhancing both attention and appraisal components within the emotional-regulation process. Trial registration: ClinicalTrials.gov, NCT05583695.

Novel Genetic and Phenotypic Expansion in GOSR2-Related Progressive Myoclonus Epilepsy.

Biallelic variants in the Golgi SNAP receptor complex member 2 gene (GOSR2) have been reported in progressive myoclonus epilepsy with neurodegeneration. Typical clinical features include ataxia and areflexia during early childhood, followed by seizures, scoliosis, dysarthria, and myoclonus. Here, we report two novel patients from unrelated families with a GOSR2-related disorder and novel genetic and clinical findings. The first patient, a male compound heterozygous for the GOSR2 splice site variant c.336+1G>A and the novel c.364G>A,p.Glu122Lys missense variant showed global developmental delay and seizures at the age of 2 years, followed by myoclonus at the age of 8 years with partial response to clonazepam. The second patient, a female homozygous for the GOSR2 founder variant p.Gly144Trp, showed only mild fine motor developmental delay and generalized tonic-clonic seizures triggered by infections during adolescence, with seizure remission on levetiracetam. The associated movement disorder progressed atypically slowly during adolescence compared to its usual speed, from initial intention tremor and myoclonus to ataxia, hyporeflexia, dysmetria, and dystonia. These findings expand the genotype-phenotype spectrum of GOSR2-related disorders and suggest that GOSR2 should be included in the consideration of monogenetic causes of dystonia, global developmental delay, and seizures.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

A Comprehensive Assessment Protocol for Swallowing (CAPS): Paving the Way towards Computer-Aided Dysphagia Screening.

Dysphagia is one of the most common problems among older adults, which might lead to aspiration pneumonia and eventual death. It calls for a feasible, reliable, and standardized screening or assessment method to prompt rehabilitation measures and mitigate the risks of dysphagia complications. Computer-aided screening using wearable technology could be the solution to the problem but is not clinically applicable because of the heterogeneity of assessment protocols. The aim of this paper is to formulate and unify a swallowing assessment protocol, named the Comprehensive Assessment Protocol for Swallowing (CAPS), by integrating existing protocols and standards. The protocol consists of two phases: the pre-test phase and the assessment phase. The pre-testing phase involves applying different texture or thickness levels of food/liquid and determining the required bolus volume for the subsequent assessment. The assessment phase involves dry (saliva) swallowing, wet swallowing of different food/liquid consistencies, and non-swallowing (e.g., yawning, coughing, speaking, etc.). The protocol is designed to train the swallowing/non-swallowing event classification that facilitates future long-term continuous monitoring and paves the way towards continuous dysphagia screening.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Cardiovascular Risk Profile and Osteoarthritis-Considering Sex and Multisite Joint Involvement: A Canadian Longitudinal Study on Aging.

OBJECTIVE: The objective of this study was to investigate a profile of cardiovascular disease (CVD) risk factors by sex among individuals with and without osteoarthritis (OA) and to consider single-site and multisite joint OA. METHODS: Data were sourced from Cycle 1, Comprehensive Cohort, Canadian Longitudinal Study on Aging, a national sample of individuals ages 45 to 85 years. Systemic inflammatory/metabolic CVD risk factors collected were high-sensitivity C-reactive protein (hsCRP) level, high-density lipoprotein, triglycerides, total cholesterol, body mass index (BMI), systolic blood pressure, and hemoglobin A1c. Smoking history was also collected. Respondents indicated doctor-diagnosed OA in the knees, hips, and/or hands and were characterized as yes/no OA and single site/multisite OA. Individuals with OA were age- and sex-matched to non-OA controls. Covariates were age, sex, education, income, physical activity, timed up and go test findings, and comorbidities. A latent CVD risk variable was derived in women and men; standardized scores were categorized as follows: lowest, mid-low, mid-high, and highest risk. Associations with OA were quantified using ordinal logistic regressions. RESULTS: A total of 6,098 respondents (3,049 with OA) had a median age of 63 years, and 55.8% were women. One-third of OA respondents were in the highest risk category versus one-fifth of non-OA respondents. Apart from BMI (the largest contributor in both sexes), hsCRP level (an inflammation marker) was predominant in women, and metabolic factors and smoking were predominant in men. Overall, OA was associated with worse CVD risk quartiles compared with non-OA. OA was increasingly associated with worse CVD risk quartiles with increasing risk thresholds among women with multisite OA, but not men. CONCLUSION: Findings suggest unique CVD risks by sex/multisite subgroups and point to a potentially important role for inflammation in OA over and above traditional CVD risk factors.

Biochemical Characterization of the TINTIN Module of the NuA4 Complex Reveals Allosteric Regulation of Nucleosome Interaction.

Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.

A balancing act: interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans.

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1's role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity.

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein.

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.

Conformational landscape of the yeast SAGA complex as revealed by cryo-EM.

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.

Biochemical and Structural Characterization of Human Core Elongator and Its Subassemblies.

Conserved from yeast to humans and composed of six core subunits (Elp1-Elp6), Elongator is a multiprotein complex that catalyzes the modification of the anticodon loop of transfer RNAs (tRNAs) and in turn regulates messenger RNA decoding efficiency. Previous studies showed that yeast Elongator consists of two subassemblies (yElp1/2/3 and yElp4/5/6) and adopts an asymmetric overall architecture. Yet, much less is known about the structural properties of the orthologous human Elongator. Furthermore, the order in which the different Elongator subunits come together to form the full assembly as well as how they coordinate with one another to catalyze tRNA modification is not fully understood. Here, we purified recombinant human Elongator subunits and subassemblies and examined them by single-particle electron microscopy. We found that the human Elongator complex is assembled from two subcomplexes that share similar overall morphologies as their yeast counterparts. Complementary co-purification and pulldown assays revealed that the scaffolding subunit human ELP1 (hELP1) has stabilizing effects on the human ELP3 catalytic subunit. Furthermore, the peripheral hELP2 subunit appears to enhance the integrity and substrate-binding ability of the dimeric hELP1/2/3. Lastly, we found that hELP4/5/6 is recruited to hELP1/2/3 via hELP3. Collectively, our work generated insights into the assembly process of core human Elongator and the coordination of different subunits within this complex.

Swallow Detection with Acoustics and Accelerometric-Based Wearable Technology: A Scoping Review.

Swallowing disorders, especially dysphagia, might lead to malnutrition and dehydration and could potentially lead to fatal aspiration. Benchmark swallowing assessments, such as videofluoroscopy or endoscopy, are expensive and invasive. Wearable technologies using acoustics and accelerometric sensors could offer opportunities for accessible and home-based long-term assessment. Identifying valid swallow events is the first step before enabling the technology for clinical applications. The objective of this review is to summarize the evidence of using acoustics-based and accelerometric-based wearable technology for swallow detection, in addition to their configurations, modeling, and assessment protocols. Two authors independently searched electronic databases, including PubMed, Web of Science, and CINAHL. Eleven (n = 11) articles were eligible for review. In addition to swallowing events, non-swallowing events were also recognized by dry (saliva) swallowing, reading, yawning, etc., while some attempted to classify the types of swallowed foods. Only about half of the studies reported that the device attained an accuracy level of >90%, while a few studies reported poor performance with an accuracy of <60%. The reviewed articles were at high risk of bias because of the small sample size and imbalanced class size problem. There was high heterogeneity in assessment protocol that calls for standardization for swallowing, dry-swallowing and non-swallowing tasks. There is a need to improve the current wearable technology and the credibility of relevant research for accurate swallowing detection before translating into clinical screening for dysphagia and other swallowing disorders.

Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation.

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex.

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.