ATF6 protects against protein misfolding during cardiac hypertrophy.

Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.

The proteostasis interactomes of trafficking-deficient K V 11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue.

Introduction: The voltage gated potassium ion channel K V 11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery, comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. Methods: We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) K V 11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. Resultsa: We identified 573 core K V 11.1 protein interactors. Both variants K V 11.1-G601S and K V 11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for K V 11.1 degradation and that the K V 11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of K V 11.1 in regulating trafficking. Conclusion: Our report characterizes the proteostasis network of K V 11.1, two trafficking deficient K V 11.1 variants, and variants treated with a pharmacological chaperone. The identified protein interactions could be targeted therapeutically to improve K V 11.1 trafficking and treat Long QT Syndrome.

Benchmarking AlphaMissense pathogenicity predictions against cystic fibrosis variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis-a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro-demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.

Expression of SARS-CoV-2 Nonstructural Proteins 3 and 4 Can Tune the Unfolded Protein Response in Cell Culture.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through the activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of overexpression of SARS-CoV-2 nsp4, a key driver of replication, on the UPR in cell culture using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find that nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found that an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their coexpression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Reanalysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.

Benchmarking AlphaMissense Pathogenicity Predictions Against Cystic Fibrosis Variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis - a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro - demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.

Site-specific crosslinking reveals Phosphofructokinase-L inhibition drives self-assembly and attenuation of protein interactions.

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo, respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

Site-Specific Crosslinking Reveals Phosphofructokinase-L Inhibition Drives Self-Assembly and Attenuation of Protein Interactions.

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

CFTR Folding: From Structure and Proteostasis to Cystic Fibrosis Personalized Medicine.

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations in the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR). Class-II mutants of CFTR lack intermolecular interactions important for CFTR structural stability and lead to misfolding. Misfolded CFTR is detected by a diverse suite of proteostasis factors that preferentially bind and route mutant CFTR toward premature degradation, resulting in reduced plasma membrane CFTR levels and impaired chloride ion conductance associated with CF. CF treatment has been vastly improved over the past decade by the availability of small molecules called correctors. Correctors directly bind CFTR, stabilize its structure by conferring thermodynamically favorable interactions that compensate for mutations, and thereby lead to downstream folding fidelity. However, each of over 100 Class-II CF causing mutations causes unique structural defects and shows a unique response to drug treatment, described as theratype. Understanding CFTR structural defects, the proteostasis factors evaluating those defects, and the stabilizing effects of CFTR correctors will illuminate a path toward personalized medicine for CF. Here, we review recent advances in our understanding of CFTR folding, focusing on structure, corrector binding sites, the mechanisms of proteostasis factors that evaluate CFTR, and the implications for CF personalized medicine.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, a key driver of replication, on the UPR using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their co-expression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Re-analysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.

General trends in the effects of VX-661 and VX-445 on the plasma membrane expression of clinical CFTR variants.

Cystic fibrosis (CF) is caused by mutations that compromise the expression and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Most people with CF harbor a common misfolded variant (ΔF508) that can be partially rescued by therapeutic "correctors" that restore its expression. Nevertheless, many other CF variants are insensitive to correctors. Using deep mutational scanning, we quantitatively compare the effects of two correctors on the plasma membrane expression of 129 CF variants. Though structural calculations suggest corrector binding provides similar stabilization to most variants, it's those with intermediate expression and mutations near corrector binding pockets that exhibit the greatest response. Deviations in sensitivity appear to depend on the degree of variant destabilization and the timing of misassembly. Combining correctors appears to rescue more variants by doubling the binding energy and stabilizing distinct cotranslational folding transitions. These results provide an overview of rare CF variant expression and establish new tools for precision pharmacology.

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis.

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol-requiring enzyme-1 (IRE1) operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-box binding protein 1 (XBP1). To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in the brain. Overexpression of XBP1 in the nervous system using transgenic mice reduced the load of amyloid deposits and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an 5xFAD mice using adeno-associated vectors improved different AD features. XBP1 expression corrected a large proportion of the proteomic alterations observed in the AD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3, a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased in H3K4me1 levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels, but also causes a decrease in H3K4me3 levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific demethylase 5 (KDM5/Lid), an enzyme that removes tri- and di- methyl marks from H3K4. Moreover, analysis of ChIP-seq data revealed that BRWD3 and KDM5 are significantly co- localized throughout the genome and that sites of H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

The histone chaperone NASP maintains H3-H4 reservoirs in the early Drosophila embryo.

Histones are essential for chromatin packaging, and histone supply must be tightly regulated as excess histones are toxic. To drive the rapid cell cycles of the early embryo, however, excess histones are maternally deposited. Therefore, soluble histones must be buffered by histone chaperones, but the chaperone necessary to stabilize soluble H3-H4 pools in the Drosophila embryo has yet to be identified. Here, we show that CG8223, the Drosophila homolog of NASP, is a H3-H4-specific chaperone in the early embryo. We demonstrate that, while a NASP null mutant is viable in Drosophila, NASP is a maternal effect gene. Embryos laid by NASP mutant mothers have a reduced rate of hatching and show defects in early embryogenesis. Critically, soluble H3-H4 pools are degraded in embryos laid by NASP mutant mothers. Our work identifies NASP as the critical H3-H4 histone chaperone in the Drosophila embryo.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knock-downs sensitize P67L to VX-445 and further enhance the correction of this variant. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.

Benchmarking AlphaFold2 on peptide structure prediction.

Recent advancements in computational tools have allowed protein structure prediction with high accuracy. Computational prediction methods have been used for modeling many soluble and membrane proteins, but the performance of these methods in modeling peptide structures has not yet been systematically investigated. We benchmarked the accuracy of AlphaFold2 in predicting 588 peptide structures between 10 and 40 amino acids using experimentally determined NMR structures as reference. Our results showed AlphaFold2 predicts α-helical, ß-hairpin, and disulfide-rich peptides with high accuracy. AlphaFold2 performed at least as well if not better than alternative methods developed specifically for peptide structure prediction. AlphaFold2 showed several shortcomings in predicting Φ/Ψ angles, disulfide bond patterns, and the lowest RMSD structures failed to correlate with lowest pLDDT ranked structures. In summary, computation can be a powerful tool to predict peptide structures, but additional steps may be necessary to analyze and validate the results.

Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging.

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

Compendium of proteins containing segments that exhibit zero-tolerance to amino acid variation in humans.

Genetic missense tolerance ratio (MTR) analysis systematically evaluates all possible segments in a given protein-encoding transcript found in the human population. This method scores each segment for the number of observed missense variants versus the number of silent mutations in that same segment. An MTR score of 0 indicates that no missense mutations are observed within a given segment. This is indicative of evolutionary purifying selection, which excludes mutations in that segment from the general human population. Here, we conducted MTR analysis on each of the roughly 20,000 protein-encoding human genes. It was seen that there are 257 genes with at least one 31-residue encoding segment with MTR = 0 (1.3% of all human genes). The proteins encoded by these 257 genes were tabulated along with information regarding the sequence location of each intolerant segment, the likely function of the protein, and so forth. The most functionally-enriched family among these proteins is a collection of several dozen proteins that are directly involved in RNA splicing. Some of the other proteins with zero-tolerance segments have thus far escaped significant characterization. Indeed, while a number of these proteins have previously been genetically linked to human disorders, many have not. We hypothesize that this compendium of human proteins with zero-tolerance segments can be used to complement disease mutation data as a pointer to genes and proteins that are associated with interesting and underexplored human biology.