Disease-associated Mutations Research Articles

Computational Study of the Activation Mechanism of Wild-Type Parkin and Its Clinically Relevant Mutant.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. It impairs the control of movement and balance. Parkin mutations worsen the symptoms in sporadic cases and cause the early onset of the disease. Therefore, recent efforts have focused on the rescue of defective parkin by engineered proteins or small-molecule activators to enhance parkin activation. These attempts require holistic understanding of the multistep activation mechanism and molecular effects of disease-associated mutations. Hereby, we provided a comprehensive analysis of the activation mechanism of parkin and a clinically relevant mutant, parkinS167N, using molecular dynamics simulations based on the following crystal structures: (1) parkin, (2) parkin/pUb (phosphorylated Ubiquitin), (3) pparkin/pUb, and (4) pparkin/pUb/UbcH7-Ub. Each of these represents an individual step in the activation process. We showed that the mutation impacted the dynamics of not only the RING0 domain, where it is localized, but also the RING2, Ubl, and IBR domains. We identified residues participating in the allosteric interaction network involved in parkin activation. Some of them are mutated in PD-associated parkin variants. The RING0 domain provides a binding interface with various proteins, so understanding problems associated with the mutation paves the way to the discovery of effective engineered proteins or small molecules that activate mutant parkin.

Arrhythmogenic calmodulin variants D131E and Q135P disrupt interaction with the L-type voltage-gated Ca2+ channel (Cav1.2) and reduce Ca2+-dependent inactivation.

Long QT syndrome (LQTS) and catecholaminergic polymorphism ventricular tachycardia (CPVT) are inherited cardiac disorders often caused by mutations in ion channels. These arrhythmia syndromes have recently been associated with calmodulin (CaM) variants. Here, we investigate the impact of the arrhythmogenic variants D131E and Q135P on CaM's structure-function relationship. Our study focuses on the L-type calcium channel Cav1.2, a crucial component of the ventricular action potential and excitation-contraction coupling. We used circular dichroism (CD), 1H-15N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca2+ and interaction of Ca2+/CaM with Cav1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Cav1.2 activity was determined using HEK293-Cav1.2 cells (B'SYS) and whole-cell patch-clamp electrophysiology. Using a combination of protein biophysics and structural biology, we show that the disease-associated mutations D131E and Q135P mutations alter apo/CaM structure and stability. In the Ca2+-bound state, D131E and Q135P exhibited reduced Ca2+ binding affinity, significant structural changes, and altered interaction with Cav1.2 domains (increased affinity for Cav1.2-IQ and decreased affinity for Cav1.2-NSCaTE). We show that the mutations dramatically impair Ca2+-dependent inactivation (CDI) of Cav1.2, which would contribute to abnormal Ca2+ influx, leading to disrupted Ca2+ handling, characteristic of cardiac arrhythmia syndromes. These findings provide insights into the molecular mechanisms behind arrhythmia caused by calmodulin mutations, contributing to our understanding of cardiac syndromes at a molecular and cellular level.

Chaperone-Mediated Heterotypic Phase Separation Prevents the Amyloid Formation of the Pathological Y145Stop Prion Protein Variant.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that arrests the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

Connecting genotype and phenotype in minor spliceosome diseases

Minor spliceosome is responsible for recognizing and excising a specific subset of divergent introns during the pre-mRNA splicing process. Mutations in the unique snRNA and protein components of the minor spliceosome are increasingly being associated with a variety of germline and somatic human disorders, collectively termed as minor spliceosomopathies. Understanding the mechanistic basis of these diseases has been challenging due to limited functional information on many minor spliceosome components. However, recently published cryo-electron microscopy (cryo-EM) structures of various minor spliceosome assembly intermediates have marked a significant advancement in elucidating the roles of these components during splicing. These structural breakthroughs have not only enhanced our comprehension of the minor spliceosome’s functionality but also shed light on how disease-associated mutations disrupt its functions. Consequently, research focus is now shifting toward investigating how these splicing defects translate into broader pathological processes within gene expression pathways. Here we outline the current structural and functional knowledge of the minor spliceosome, explore the mechanistic consequences of its mutations, and discuss emerging challenges in connecting molecular dysfunctions to clinical phenotypes.

The topography of nullomer-emerging mutations and their relevance to human disease.

Nullomers are short DNA sequences (11-18 base pairs) that are absent from a genome; however, they can emerge due to mutations. Here, we characterize all possible putative human nullomer-emerging single base pair mutations, population variants and disease-causing mutations. We find that the primary determinants of nullomer emergence in the human genome are the presence of CpG dinucleotides and methylated cytosines. Putative nullomer-emerging mutations are enriched at specific genomic elements, including transcription start and end sites, splice sites and transcription factor binding sites. We also observe that putative nullomer-emerging mutations are more frequent in highly conserved regions and show preferential location at nucleosomes. Among repeat elements, Alu repeats exhibit pronounced enrichment for putative nullomer-emerging mutations at specific positions. Finally, we find that disease-associated pathogenic mutations are significantly more likely to cause emergence of nullomers than their benign counterparts.

SCREEN: A Graph-based Contrastive Learning Tool to Infer Catalytic Residues and Assess Enzyme Mutations.

when applied to inferred enzyme structures; and (iii) generalizes well to enzymes dissimilar from those in the training set. We also show that the putative catalytic residues predicted by SCREEN mimic key structural and biophysical characteristics of native catalytic residues. Moreover, using experimental data sets, we show that SCREEN's predictions can be used to distinguish residues with a high mutation tolerance from those likely to cause functional loss when mutated, indicating that this tool might be used to infer disease-associated mutations. SCREEN is publicly available at https://github.com/BioColLab/SCREEN and https://ngdc.cncb.ac.cn/biocode/tool/7580.

Identification of polycystin 2 missense mutants targeted for endoplasmic reticulum-associated degradation.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder leading to end-stage renal disease. ADPKD arises from mutations in the PKD1 and PKD2 genes, which encode polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC2 is a nonselective cation channel, and disease-linked mutations disrupt normal cellular processes, including signaling and fluid secretion. In this study, we investigate whether disease-causing missense mutations compromise PC2 folding, an event that can lead to endoplasmic reticulum-associated degradation (ERAD). To this end, we first developed a new yeast PC2 expression system. We show that the yeast system provides a tractable model to investigate PC2 biogenesis and that a disease-associated PC2 mutant, D511V, exhibits increased polyubiquitination and accelerated proteasome-dependent degradation compared with wild-type PC2. In contrast to wild-type PC2, the PC2 D511V variant also failed to improve the growth of yeast strains that lack endogenous potassium transporters, highlighting a loss of channel function at the cell surface and a new assay for loss-of-function PKD2 variants. In HEK293 cells, both D511V along with another disease-associated mutant, R322Q, were targeted for ERAD. Consistent with defects in protein folding, the surface localization of these PC2 variants was increased by incubation at low-temperature in HEK293 cells, underscoring the potential to pharmacologically rescue these and perhaps other misfolded PC2 alleles. Together, our study supports the hypothesis that select PC2 missense variants are degraded by ERAD, the potential for screening PKD2 alleles in a new genetic system, and the possibility that chemical chaperone-based therapeutic interventions might be used to treat ADPKD.NEW & NOTEWORTHY This study indicates that select missense mutations in PC2, a protein that when mutated leads to ADPKD, result in protein misfolding and degradation via the ERAD pathway. Our work leveraged a new yeast model and an HEK293 cell model to discover the mechanism underlying PC2 instability and demonstrates the potential for pharmacological rescue. We also suggest that targeting the protein misfolding phenotype with chemical chaperones may offer new therapeutic strategies to manage ADPKD-related protein dysfunction.

Hsp90 and cochaperones have two genetically distinct roles in regulating eEF2 function.

Protein homeostasis relies on the accurate translation and folding of newly synthesized proteins. Eukaryotic elongation factor 2 (eEF2) promotes GTP-dependent translocation of the ribosome during translation. eEF2 folding was recently shown to be dependent on Hsp90 as well as the cochaperones Hgh1, Cns1, and Cpr7. We examined the requirement for Hsp90 and cochaperones more closely and found that Hsp90 and cochaperones have two distinct roles in regulating eEF2 function. Yeast expressing one group of Hsp90 mutations or one group of cochaperone mutations had reduced steady-state levels of eEF2. The growth of Hsp90 mutants that affected eEF2 accumulation was also negatively affected by deletion of the gene encoding Hgh1. Further, mutations in yeast eEF2 that mimic disease-associated mutations in human eEF2 were negatively impacted by loss of Hgh1 and growth of one mutant was partially rescued by overexpression of Hgh1. In contrast, yeast expressing different groups of Hsp90 mutations or a different cochaperone mutation had altered sensitivity to diphtheria toxin, which is dictated by a unique posttranslational modification on eEF2. Our results provide further evidence that Hsp90 contributes to proteostasis not just by assisting protein folding, but also by enabling accurate translation of newly synthesized proteins. In addition, these results provide further evidence that yeast Hsp90 mutants have distinct in vivo effects that correlate with defects in subsets of cochaperones.

Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.

Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.

Quantitative Proteomic Analysis of APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder affecting the central nervous system (CNS), with its etiology still shrouded in uncertainty. The interplay of extracellular amyloid-β (Aβ) deposition, intracellular neurofibrillary tangles (NFTs) composed of tau protein, cholinergic neuronal impairment, and other pathogenic factors is implicated in the progression of AD. The current study endeavors to delineate the proteomic landscape alterations in the hippocampus of an AD murine model, utilizing proteomic analysis to identify key physiological and pathological shifts induced by the disease. This endeavor aims to shed light on the underlying pathogenic mechanisms, which could facilitate early diagnosis and pave the way for novel therapeutic interventions for AD. To dissect the proteomic perturbations induced by Aβ and Presenilin-1 (PS1) in the AD pathogenesis, we undertook a label-free quantitative (LFQ) proteomic analysis focusing on the hippocampal proteome of the APP/PS1 transgenic mouse model. Employing a multi-faceted approach that included differential protein functional enrichment, cluster analysis, and protein-protein interaction (PPI) network analysis, we conducted a comprehensive comparative proteomic study between APP/PS1 transgenic mice and their wild-type C57BL/6 counterparts. Mass spectrometry identified a total of 4817 proteins in the samples, with 2762 proteins being quantifiable. Comparative analysis revealed 396 proteins with differential expression between the APP/PS1 and control groups. Notably, 35 proteins exhibited consistent temporal regulation trends in the hippocampus, with concomitant alterations in biological pathways and PPI networks. This study presents a comparative proteomic profile of transgenic (APP/PS1) and wild-type mice, highlighting the proteomic divergences. Furthermore, it charts the trajectory of proteomic changes in the AD mouse model across the developmental stages from 2 to 12 months, providing insights into the physiological and pathological implications of the disease-associated genetic mutations.

Lysosomal damage triggers a p38 MAPK-dependent phosphorylation cascade to promote lysophagy via the small heat shock protein HSP27.

Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27.

Disease-Associated Q159X Mutant Prion Protein Is Sufficient to Cause Fatal Degenerative Disease in Mice.

PRNP Q160X is one of the five dominantly inheritable nonsense mutations causing familial prion diseases. Till now, it remains unclear how this type of nonsense mutations causes familial prion diseases with unique clinical and pathological characteristics. Human prion protein (PrP) Q160X mutation is equivalent to Q159X in mouse PrP, which produces the mutant fragment PrP1-158. Through intracerebroventricular injection of recombinant adeno-associated virus in newborn mice, we successfully overexpressed mouse PrP1-158-FLAG in the central nervous system. Interestingly, high level PrP1-158-FLAG expression in the brain caused death in these mice with an average survival time of 60 ± 9.1 days. Toxicity correlated with levels of PrP1-158-FLAG but was independent of endogenous PrP. Histopathological analyses showed microgliosis and astrogliosis in mouse brains expressing PrP1-158-FLAG and most of PrP1-158-FLAG staining appeared intracellular. Biochemical characterization revealed that the majority of PrP1-158-FLAG were insoluble and a significant part of PrP1-158-FLAG appeared to contain an un-cleaved signal peptide that may contribute to its cytoplasmic localization. Importantly, an ~10-kDa proteinase K-resistant PrP fragment was detected, which was the same as those observed in patients suffering from this type of prion diseases. To our knowledge, this is the first animal study of familial prion disease caused by Q159X that recapitulates key features of human disease. It will be a valuable tool for investigating the pathogenic mechanisms underlying familial prion diseases caused by nonsense mutations.

GPTrans: A Biological Language Model-Based Approach for Predicting Disease-Associated Mutations in G Protein-Coupled Receptors.

Accurately predicting mutations in G protein-coupled receptors (GPCRs) is critical for advancing disease diagnosis and drug discovery. In response to this imperative, GPTrans has emerged as a highly accurate predictor of disease-related mutations in GPCRs. The core innovation of GPTrans resides in the design of a novel feature extraction network, that is capable of integrating features from both wildtype and mutant protein variant sites, utilizing multifeature connections within a transformer framework to ensure comprehensive feature extraction. A key aspect of GPTrans's effectiveness is our introduction of an innovative deep feature integration strategy, which merges embeddings and class tokens from multiple protein language models, including evolutionary scale modeling and ProtTrans, thus shedding light on the biochemical properties of proteins. Leveraging transformer components and a self-attention mechanism, GPTrans captures higher-level representations of protein features. Employing both wildtype and mutation site information for feature fusion not only enriches the predictive feature set but also avoids the common issue of overestimation associated with sequence-based predictions. This approach distinguishes GPTrans, enabling it to significantly outperform existing methods. Our evaluations across diverse GPCR data sets, including ClinVar and MutHTP, demonstrate GPTrans's superior performance, with average AUC values of 0.874 and 0.590 in 10-fold cross-validation. Notably, compared to the AlphaMissense method, GPTrans exhibited a remarkable 38.03% improvement in accuracy when predicting disease-associated mutations in the MutHTP data set. A thorough analysis of the predicted results further validates the model's effectiveness. The source code, data sets, and prediction results for GPTrans are available for academic use at https://github.com/EduardWang/GPTrans.

Structural basis for the reaction cycle and transport mechanism of human Na+-sulfate cotransporter NaS1 (SLC13A1).

Sulfate (SO42-) is a pivotal inorganic anion with essential roles in mammalian physiology. NaS1, a member of solute carrier 13 family and divalent anion/sodium symporter family, functions as a Na+-sulfate cotransporter, facilitating sulfate (re)absorption across renal proximal tubule and small intestine epithelia. While previous studies have linked several human disorders to mutations in the NaS1 gene, its transport mechanism remains unclear. Here, we report the cryo-electron microscopy structures of five distinct conformations of the human NaS1 at resolutions of 2.7 to 3.3 angstroms, revealing the substrates recognition mechanism and the conformational change of NaS1 during the Na+-sulfate cotransport cycle. Our studies delineate the molecular basis of the detailed dynamic transport cycle of NaS1. These findings advance the current understanding of the Na+-sulfate cotransport mechanism, human sulfate (re)absorption, and the implications of disease-associated NaS1 mutations.

The effect of a dominant kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy on brain development and neuropathology

Amino acid substitutions in the kinase domain of the human CSF1R protein are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Previous analysis demonstrated that heterozygous mutation (Csf1rE631K/+) had a dominant inhibitory effect on CSF1R signaling in vitro and in vivo but did not recapitulate human disease pathology. We speculated that leukoencephalopathy in humans requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles. Here we examine the Csf1rE631K/+ mutation impact on microglial phenotype, postnatal brain development, age-related changes in gene expression and on prion disease and experimental autoimmune encephalitis (EAE), two pathologies in which microgliosis is a prominent feature. The Csf1rE631K/+ mutation reduced microglial abundance and the expression of microglial-associated transcripts relative to wild-type controls at 12 and 43 weeks of age. There was no selective effect on homeostatic markers e.g. P2ry12, or age-related changes in gene expression in striatum and hippocampus. An epistatic interaction was demonstrated between Csf1rE631K/+ and Cx3cr1EGFP/+ genotypes leading to dysregulated microglial and neuronal gene expression in hippocampus and striatum. Heterozygous Csf1rE631K mutation reduced the microgliosis associated with both diseases. There was no significant impact on disease severity or progression in prion disease. In EAE, inflammation-associated transcripts in the hippocampus and striatum were suppressed in parallel with microglia-specific transcripts. The results support a dominant inhibitory model of CSF1R-related leukoencephalopathy and likely contributions of an environmental trigger and/or genetic background to neuropathology.

Cancer-associated DAXX mutations reveal a critical role for ATRX localization in ALT suppression.

To maintain genome stability, proliferating cells must enact a program of telomere maintenance. While most tumors maintain telomeres through the action of telomerase, a subset of tumors utilize a DNA-templated process termed Alternative Lengthening of Telomeres or ALT. ALT is associated with mutations in the ATRX/DAXX/H3.3 histone chaperone complex, which is responsible for deposition of non-replicative histone variant H3.3 at heterochromatic regions of the genome including telomeres. We wished to better understand the role DAXX plays in ALT suppression, and to determine which disease-associated DAXX mutations are unable to suppress ALT. To answer this question, we have leveraged the G292 cell line, in which ATRX is wild type but DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. Restoration of wild-type DAXX in G292 localizes ATRX and abrogates ALT. Using this model system, we tested the ability of a panel of disease-associated DAXX missense variants to suppress ALT. Missense mutations in the ATRX binding domain, the histone binding domain, and the C-terminal SUMO interaction motif reduce the ability of DAXX to suppress ALT. Unexpectedly, we find that mutations in the DAXX histone binding domain lead to failure of ATRX localization. We conclude that a key function of DAXX in ALT suppression is the localization of ATRX to nuclear foci.

Mutations in Mig6 reduce inhibition of the epidermal growth factor receptor.

Mitogen-inducible gene 6 (Mig6) is a cellular inhibitor of epidermal growth factor receptor (EGFR) that binds directly to the EGFR kinase domain and interferes with signaling. Reduced Mig6 expression is correlated with increased EGFR activity in multiple cancer models. Here, we investigated whether disease-associated point mutations could reduce the inhibitory potency of Mig6. We show that several cancer-associated mutations, and a mutation derived from Alzheimer's Disease patients, diminish the ability of Mig6 to bind and inhibit EGFR invitro. In mammalian cells, the mutations decreased the Mig6-induced suppression of basal and EGF-stimulated autophosphorylation, MAP kinase phosphorylation, and cell migration. To probe the mechanisms by which the mutations could lead to reduced Mig6 inhibition, we constructed atomic-level computational models of Mig6 complexed with the EGFR catalytic domain, and performed molecular dynamics simulations for wild-type and mutant complexes.

INF2 mutations cause kidney disease through a gain-of-function mechanism.

Heterozygosity for inverted formin-2 (INF2) mutations causes focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth disease. A key question is whether the disease is caused by gain-of-function effects on INF2 or loss of function (haploinsufficiency). Despite established roles in multiple cellular processes, neither INF2 knockout mice nor mice with a disease-associated point mutation display an evident kidney or neurologic phenotype. Here, we compared responses to puromycin aminonucleoside (PAN)-induced kidney injury between INF2 R218Q and INF2 knockout mice. R218Q INF2 mice are susceptible to glomerular disease, in contrast to INF2 knockout mice. Colocalization, coimmunoprecipitation analyses, and cellular actin measurements showed that INF2 R218Q confers a gain-of-function effect on the actin cytoskeleton. RNA expression analysis showed that adhesion and mitochondria-related pathways were enriched in the PAN-treated R218Q mice. Both podocytes from INF2 R218Q mice and human kidney organoids with an INF2 mutation (S186P) recapitulate adhesion and mitochondrial phenotypes. Thus, gain-of-function mechanisms drive INF2-related FSGS and explain this disease's autosomal dominant inheritance.

The N-terminal region of DNMT3A engages the nucleosome surface to aid chromatin recruitment

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.

Conformational plasticity links structural instability of NAA10F128I and NAA10F128L mutants to their catalytic deregulation

The acetylation of proteins' N-terminal amino groups by the N-acetyltransferase complexes plays a crucial role in modulating the spatial stability and functional activities of diverse human proteins. Mutations disrupting the stability and function of NAA10 result in X-linked rare genetic disorders. In this study, we conducted a global analysis of the impact of fifteen disease-associated missense mutations in NAA10. The analyses revealed that mutations in specific residues, such as Y43, V107, V111, and F128, predictably disrupted interactions essential for NAA10 stability, while most mutations (except R79C, A111W, Q129P, and N178K) expectedly led to structural destabilization. Mutations in many conserved residues within short linear motifs and post-translational modification sites were predicted to affect NAA10 functionality and regulation. All mutations were classified as pathogenic, with F128I and F128L identified as the most destabilizing mutations. The findings show that the F128L and F128I mutations employ different mechanisms for the loss of catalytic activities of NAA10F128L and NAA10F128I due to their structural instability. These two mutations induce distinct folding energy states that differentially modulate the structures of different regions of NAA10F128L and NAA10F128I. Specifically, the predicted instability caused by the F128I mutation results in decreased flexibility within the substrate-binding region, impairing the substrate peptide binding ability of NAA10F128I. Conversely, F128L is predicted to reduce the flexibility of the region containing the acetyl-CoA binding residues in NAA10F128L. Our study provides insights into the mechanism of catalytic inactivation of mutants of NAA10, particularly elucidating the mechanistic features of the structural and functional pathogenicity of the F128L and F128I mutations.