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At least 19 recordsLinked to original sources
PubMed2025

A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality.

Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism explains why, in the mass spectrometry data, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest widespread NCLE misfolding, that such misfolded states in non-essential proteins can bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.

Journal Article↗
PubMed2026

Disruption of the ubiquitin-mediated proteolysis pathway: a study of seed aging in Saposhnikovia divaricata caused by UBC1 gene family suppression.

BACKGROUND: Saposhnikovia divaricata (Turcz.) Schischk. is a perennial herb whose seed aging during storage significantly reduces germination rates, limiting industrial-scale production. Reactive oxygen species (ROS)-induced oxidative damage is a key driver of seed aging, but the underlying mechanisms in Saposhnikovia divaricata remain unclear. RESULTS: Suppression of the UBC1 gene family reduces the activity of ubiquitin-conjugating enzymes, leading to dysfunction of the ubiquitin-mediated proteolysis pathway, which in turn decreases protein degradation efficiency and causes the accumulation of damaged proteins. Transcriptome analysis revealed predominant downregulation of genes crucial for seed physiological maintenance. By the fourth year of storage, germination dropped sharply to 30.67%, accompanied by embryo cavitation. Downregulation of ribosome pathway genes hindered ribosome assembly and protein synthesis, while suppression of endoplasmic reticulum protein processing genes led to unfolded/misfolded protein accumulation and intensified cellular stress, accelerating aging. Proteomic analysis showed increased total differential and antioxidant-related proteins. ROS content fluctuated with storage time: peroxyl radicals peaked in year two (5.68 RFU/mg), whereas hydroxyl radicals and hydrogen peroxide were highest in year four (0.0655 pg/mL and 0.0946 pg/mL, respectively), with significant differences across periods. Elevated membrane-related proteins, increased electrical conductivity, and malondialdehyde content (maximum 54.30 nmol/g at year four) confirmed oxidative membrane damage. ROS-induced stress promotes protein misfolding, and reduced UBC1 expression is associated with impaired clearance of misfolded proteins by the ubiquitin-mediated proteolysis pathway. CONCLUSIONS: This study provides the first integrated transcriptomic and proteomic insight into UBC1 deficiency-mediated seed aging in Saposhnikovia divaricata. The findings enhance molecular understanding of seed aging and offer new directions for improving seed storage and viability.

Ubiquitin-Conjugating Enzymes↗
PubMed2026

Bi-allelic loss-of-function variants in JKAMP cause a neurodevelopmental syndrome associated with dysregulation of GPR37 trafficking.

The endoplasmic reticulum (ER) serves as a key hub for protein homeostasis, maintaining a strict quality-control system that ensures only properly folded proteins reach their destinations, while misfolded proteins are degraded via ER-associated degradation (ERAD) or selective ER-phagy. JKAMP, which encodes an ER-resident transmembrane protein involved in ERAD, has not previously been associated with human disease. Here, we report bi-allelic loss-of-function variants in JKAMP in 14 affected individuals from 10 unrelated families presenting with a neurodevelopmental syndrome characterized by intellectual disability, developmental delay, seizures, hypotonia, microcephaly, and dysmorphic features. An in vivo zebrafish model lacking jkamp recapitulated key aspects of the human disorder, including developmental abnormalities and impaired myelin production, further corroborating its pathogenic role. Mechanistic studies identified GPR37, a brain-enriched orphan G protein-coupled receptor (GPCR) and known JKAMP interactor, as a critical downstream effector. GPR37 plays essential roles in dopaminergic signaling, inflammatory pain regulation, neuroprotection, and myelination. Loss of JKAMP resulted in defective folding and degradation of GPR37, leading to its accumulation within the ER and impaired trafficking to the plasma membrane, likely due to impaired ER quality control. These findings establish JKAMP as a previously unrecognized contributor to human neurodevelopment and uncover a pathogenic mechanism linking ER protein quality control to GPCR regulation and neurological disease.

Humans↗
PubMed2024

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins.

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase, characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum. Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from endoplasmic reticulum-associated protein degradation in a catalytic-independent manner, promote autophagy, and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by the loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a deubiquitinase substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation, and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis," by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to the secretion of lysosomal proteins, including LGMN.

Humans↗
PubMed2026

Proteome analyses reveal endoplasmic reticulum stress-induced changes in protein abundance associated with Ube2j2 deficiency in human cell culture.

The unfolded protein response (UPR) helps reinstate cellular proteostasis upon an accumulation of misfolded proteins in the endoplasmic reticulum (ER), in part through ER-associated degradation (ERAD). Ube2j2 is an ER-localized E2 ubiquitin-conjugating enzyme that participates in ERAD. We used mass spectrometry analysis of cultured U2OS cells to investigate how the loss of Ube2j2 affects the cellular proteome in response to tunicamycin-induced ER stress. We constructed a network of twelve statistically distinct modules of protein abundance profiles across conditions. We describe the gene ontology annotations for each module along with the "hub gene" proteins whose abundance levels most closely adhere to each module's protein abundance profile. Our analysis identifies known Ube2j2-associated pathways (eg the UPR and ERAD) and cellular functions that were previously unassociated with Ube2j2 (eg RNA metabolism, ER-Golgi transport, and cell-cycle progression). These data are available via ProteomeXchange with identifier PXD076153 and provide avenues for further investigation into the cellular functions of Ube2j2 under basal and ER-stressed conditions.

Humans↗
PubMed2025

Silencing FAF2 mitigates alcohol-induced hepatic steatosis by modulating lipolysis and PCSK9 pathway.

BACKGROUND: Chronic alcohol consumption leads to lipid accumulation, oxidative stress, cellular damage, and inflammation in the liver, collectively referred to as alcohol-associated liver disease (ALD). FAF2/UBXD8/ETEA (Fas-associated factor 2) is a ubiquitin ligase adaptor protein that plays a crucial role in the ubiquitin-mediated degradation of misfolded proteins in the endoplasmic reticulum. A recent genome-wide association study indicated an association between FAF2 and ALD; however, the exact contribution of FAF2 to ALD pathogenesis remains unclear. METHODS: FAF2 was knocked down using AAV-delivered shRNA in C57/BL6 mice. Mice were subjected to a chronic-plus-single binge ethanol feeding (NIAAA) model. Nine hours after gavage, liver, blood, and other organs of interest were collected for gene expression and biochemical analyses. RESULTS: We first observed a significant elevation in hepatic FAF2 protein expression in individuals with ALD and in mice subjected to an ethanol-binge model. Interestingly, knocking down FAF2 in the liver using adeno-associated virus serotype 8-delivered short hairpin RNA conferred a protective effect against alcohol-induced liver steatosis in ethanol-binged mice. Transcriptomic analysis revealed that differentially expressed genes were enriched in multiple lipid metabolism regulation pathways. Further analysis of transcription factors regulating these differentially expressed genes suggested potential regulation by SREBP1. Several SREBP1 target genes, including Fasn, Scd1, Lpin1, and Pcsk9 (proprotein convertase subtilisin/kexin type 9), were dysregulated in the livers of ethanol-fed FAF2 knockdown mice. Additionally, Pcsk9 could be regulated through the FOXO3-SIRT6 pathway in the livers of ethanol-fed FAF2 knockdown mice, leading to increased liver low-density lipoprotein receptor expression and reduced plasma LDL cholesterol levels. Furthermore, FAF2 knockdown in mouse liver enhanced adipose triglyceride lipase lipolytic activity by upregulating the adipose triglyceride lipase activator, comparative gene identification-58, and downregulating the adipose triglyceridelipase transport inhibitor, Elmod2, contributing to the alleviation of liver steatosis. CONCLUSIONS: Our study uncovers a novel mechanism involving FAF2 in the pathogenesis of ALD.

Animals↗
PubMed2026

Designing an optimized strategy for extracellular expression of recombinant human TNF-α in Escherichia coli.

Extracellular protein expression in Escherichia coli is an elegant solution that addresses the complex issue of protein misfolding while simultaneously simplifying downstream processing steps. Human TNF-α was chosen as the target protein for export since it is a therapeutically important cytokine. Different genomic knockouts were tested for the ability to sustain and enhance protein expression, and BW25113 Δ(elaA + cysW) knockout was found to give a sustained and high level of expression. To improve secretion, various tags were tested, and the MBP tag at the N-terminal end was found to give maximum enhancement in the export of hTNF-α. Even the linker peptide was found to play a critical role in export, with the Ek linker giving the highest extracellular secretion, while the intein sequence completely blocked export. The co-expression of pSecAB, which is involved in protein transport to the periplasm, was also found to be helpful in enhancing extracellular protein titers. Interestingly, pelB performed poorly as compared to the native signal sequence of MBP, which gave better results. Culture conditions were optimized, and it was observed that growing cells in TB medium at a temperature of 25 °C, coupled with a pulse of concentrated nutrients at 24 h, led to a very high extracellular accumulation of ∼1.3 g/L of MBP-hTNF-α in shake flask culture. The protein was purified and tested using L929 cells for bioactivity. Thus, a combination of genomic and bioprocess strategies allowed us to obtain high levels of soluble and active extracellular expression of hTNF-α, making this a very attractive strategy for protein production.

E. coli↗
PubMed2026

Missense mutations in the SNCA gene: Molecular mechanisms and clinical implications.

The SNCA gene on chromosome 4 encodes the alpha-synuclein (αSyn) protein, which plays a central role in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). While αSyn has established roles in synaptic vesicle dynamics and neuronal signaling, alterations in SNCA regulation and sequence contribute to protein misfolding, aggregation, and loss of function. Alterations in secondary and tertiary structure, as well as protein aggregation, affect biochemical interactions, ultimately leading to pathogenesis. This review outlines the molecular architecture of the SNCA gene, including regulatory regions, alternative splicing, and untranslated regions that influence αSyn expression and isoform diversity. Seven missense mutations of the SNCA gene are discussed in detail from the genomic level, extending to phenotypic presentations. These missense mutations have different effects on the aggregation kinetics and fibril formation. Specific genotype-phenotype correlations are evident, with mutations such as A30P and H50Q commonly resembling idiopathic PD, E46K strongly associated with DLB, and G51D, A53T, and A53E linked to atypical parkinsonism and MSA-like syndromes. Differences in age at onset, disease progression, cognitive involvement, and response to therapy further reflect mutation-specific effects and modifying influences of allelic dosage and epigenetic regulation. Collectively, these findings emphasize the importance of SNCA genetic variation in shaping disease phenotype and progression. Improving the understanding of SNCA genotype-phenotype relationships in future studies may facilitate earlier diagnosis, refine prognostic stratification, and support the development of targeted, disease-modifying therapies for synucleinopathies.

Molecular mechanisms↗
PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (∼150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ∼20% for AlphaMissense underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular protein trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗
PubMed2026

The Sigma1 ER membrane receptor promotes structural protein folding and genome packaging of dengue virus.

Dengue virus (DENV) exploits the host endoplasmic reticulum (ER) to support viral protein translation and folding, replication, and assembly, although the identity of ER factors that promote these distinct steps during infection remain unclear. Here we demonstrate that the ER-resident Sigma1 ER membrane receptor (S1R) promotes virus structural protein folding and genome packaging of DENV during infection. Under S1R knockdown (KD), DENV infection is impaired without compromising virus translation or replication. Strikingly, EM analysis revealed that DENV particles in and secreted from S1R-depleted cells are smaller, likely because they are empty particles devoid of the vRNA genome. Biochemical experiments demonstrated that S1R binds to the prM structural protein and under S1R KD, the prM, E and C structural proteins became detergent-insoluble. Thus, without S1R, all three virus structural proteins misfold, impairing efficient genome packaging. Together, these findings identify a novel ER chaperone that supports a critical DENV infection step.

Dengue Virus↗
PubMed2024

Molecular Signatures of Neurodegenerative Diseases Identified by Proteomic and Phosphoproteomic Analyses in Aging Mouse Brain.

A central hallmark of neurodegenerative diseases is the irreversible accumulation of misfolded proteins in the brain by aberrant phosphorylation. Understanding the mechanisms underlying protein phosphorylation and its role in pathological protein aggregation within the context of aging is crucial for developing therapeutic strategies aimed at preventing or reversing such diseases. Here, we applied multi-protease digestion and quantitative mass spectrometry to compare and characterize dysregulated proteins and phosphosites in the mouse brain proteome using three different age groups: young-adult (3-4 months), middle-age (10 months), and old mice (19-21 months). Proteins associated with senescence, neurodegeneration, inflammation, cell cycle regulation, the p53 hallmark pathway, and cytokine signaling showed significant age-dependent changes in abundances and level of phosphorylation. Several proteins implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) including tau (Mapt), Nefh, and Dpysl2 (also known as Crmp2) were hyperphosphorylated in old mice brain suggesting their susceptibility to the diseases. Cdk5 and Gsk3b, which are known to phosphorylate Dpysl2 at multiple specific sites, had also increased phosphorylation levels in old mice suggesting a potential crosstalk between them to contribute to AD. Hapln2, which promotes α-synuclein aggregation in patients with PD, was one of the proteins with highest abundance in old mice. CD9, which regulates senescence through the PI3K-AKT-mTOR-p53 signaling was upregulated in old mice and its regulation was correlated with the activation of phosphorylated AKT1. Overall, the findings identify a significant association between aging and the dysregulation of proteins involved in various pathways linked to neurodegenerative diseases with potential therapeutic implications.

Animals↗
PubMed2026

A targetable dependency on nonsense-mediated decay for cellular homeostasis and immune control in small cell lung cancer.

Small cell lung cancer (SCLC) is one of the most aggressive malignancies, characterized by rapid metastatic dissemination and poor overall survival. Despite harboring excessive alterations, expectedly resulting in immunogenic neoantigens, patients with SCLC remain largely refractory to immunotherapy. We found abundant frameshift mutations in SCLC, regarded as highly immunogenic, counterbalanced by a hyperactive nonsense-mediated decay (NMD) pathway, responsible for frameshift-mRNA degradation. NMD activity correlated with tumor mutational burden (TMB) across cancers, suggesting that SCLC and other TMBhigh cancers may depend on NMD to limit the accumulation of mutation-derived byproducts in order to maintain cellular homeostasis and evade immune recognition. In TMBhigh SCLC models, inhibition of NMD impaired cell proliferation and induced ER stress-dependent apoptosis due to the accumulation of misfolded proteins. Genetic and pharmacological NMD inhibition in vivo effectively controlled TMBhigh tumor growth without overt toxicity. By integrating genome and transcriptome sequencing with MHC-I immunopeptidomics and functional in vitro and in vivo assays, we identified that NMD inhibition boosted neoantigen expression and presentation by tumor cells and increased T cell recognition, thus enhancing overall tumor immunogenicity and further improving immunotherapy efficacy in vivo. Our work shows that SCLC - as a TMBhigh cancer - relies on NMD for survival and immune escape, uncovering a novel TMB-dependent tractable vulnerability for this devastating disease.

Humans↗
PubMed2026

Characterization of a Novel BTD Hypomorphic Variant in a Patient with Complex Neurodevelopmental Delay: Resolving Actionable Metabolic Vulnerabilities Beyond Borderline Plasma Biochemistry.

Plasma biochemistry often presents significant limitations in diagnosing borderline metabolic disorders, particularly within complex neurodevelopmental phenotypes. Here, we present the clinical genomic evaluation of a six-year patient presenting with early-onset hypotonia and severe gastrointestinal complications whose newborn screening panel did not evaluate biotinidase (BTD) activity. While initial baseline plasma biochemistry yielded borderline residual BTD function (46% of the population mean), targeted sequencing identified a novel, compound heterozygous hypomorphic variant (p.Thr459Met) in trans with the common p.Asp424His allele. In vitro functional validation confirmed that p.Thr459Met induces severe protein misfolding and intracellular retention, impairing enzyme secretion. Biotin supplementation triggered a documented and favorable therapeutic improvement, establishing this borderline enzymatic background as an actionable metabolic vulnerability unmasked by chronic gastrointestinal stressors. This study underscores the critical value of functional genomic characterization over static enzymatic biomarkers to identify highly treatable metabolic components within heterogeneous clinical landscapes.

Humans↗
PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important, promiscuous divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (~150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ~20% underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗
PubMed2025

A comprehensive phylogeny of mammalian PRNP gene reveals no influence of prion misfolding propensity on the evolution of this gene.

Prion diseases are invariably fatal neurodegenerative diseases that affect some mammalian species, including humans. These diseases are caused by the misfolding of the cellular prion protein (PrPC) into a pathologic isoform (PrPSc). The prion protein is highly conserved across mammals. However, some species present lower susceptibility to prion diseases than others. This behavior is likely explained by the resistance of these animal species' prion proteins to acquire a pathological conformation. Therefore, the tertiary structure and interspecific variations encoded in the primary structure determine a PrP proneness to misfolding. For this reason, we studied the PRNP gene from a phylogenetic perspective, potentially unveiling evolutionary events related to prion diseases. We generated a database of mammalian PRNP sequences and constructed phylogenetic trees based on nucleotide sequence variations. We aligned 1146 PRNP gene sequences from 901 different mammalian species and built a PRNP gene-based phylogenetic tree. Classical phylogenetic orders tend to maintain their clustering in the PRNP gene tree. Nonetheless, the few differences found may shed some light on potential evolutionary constraints posed by prion disorders. Moreover, this phylogenetic study was combined with an in vitro misfolding study. Protein Misfolding Shaking Amplification (PMSA) was used to evaluate the tendency of many of these proteins to misfold. This comprehensive analysis spanned a wide range of mammalian prion protein sequences and included analysis of different variants with a focus on the human rs1799990 locus (c.385A > G, p.Met129Val). This variant, widely linked to prion disease susceptibility in humans, is explored in the context of its evolutionary origins. All in all, our PRNP gene-based tree, despite showing some topological differences with the reference species tree that could be in some cases related to prion disease susceptibility, is not significantly distinct. Indicating that the proneness of a PrP variant to misfold spontaneously has not shaped the evolution of this gene.

Phylogeny↗
PubMed2026

Lipid-nanoparticle-mediated base editing of the trabecular meshwork rescues glaucoma in vivo.

Mutations in MYOC, the most common genetic cause of glaucoma, cause misfolded myocilin to accumulate in the endoplasmic reticulum (ER), leading to trabecular meshwork (TM) dysfunction, elevated intraocular pressure, and progressive vision loss. While gene editing offers curative potential, current delivery methods rely on viral vectors, which are limited by inflammation, off-target effects, and poor translatability. Here, we report a nonviral lipid nanoparticle (LNP) platform that enables selective in vivo delivery of mRNA encoding an adenine base editor and single guide RNA (LNP-ABE) to TM cells. A direct comparison of LNP-mCherry with lentiviral GFP revealed that LNPs outperform viral vectors, achieving markedly higher efficiency and greater selectivity for the TM without inducing ocular inflammation. In a Cre-inducible Tg.CreMYOCY437H glaucoma mouse model, LNP-Cre mRNA selectively induced mutant MYOC expression in the TM, faithfully recapitulating key disease features. A single administration of LNP-ABE achieved efficient on-target editing of mutant MYOC, reducing mutant myocilin protein by approximately 46%, decreasing aggregates, alleviating ER stress, and fully rescuing the glaucomatous phenotype in Tg.CreMYOCY437H mice. Importantly, no off-target editing or ocular toxicity was detected. These findings establish LNP-based mRNA delivery as a safe, efficient, and clinically translatable approach for TM-targeted genome editing with broad therapeutic potential in glaucoma.

Animals↗
PubMed2025

Properties Governing Native State Entanglements and Relationships to Protein Function.

Non-covalent lasso entanglements are structural motifs found in a majority of globular proteins, and their misfolding has been linked to a range of biological consequences. Here, we characterize these motifs' structural and physicochemical properties, sequence biases, functional site correlations, and universal features across E. coli, S. cerevisiae, and H. sapiens. We find that the crossing residues, which pierce the plane of the entanglement loop, are 11-times more likely to be a β-strand than an α-helix or random coil, and that around this position the protein sequence is 2.5-times more likely to be composed of a stretch of all hydrophobic residues (most often Val, Ile, or Phe) compared to other sequence motifs. Functionally, crossing residues are enriched at enzyme active sites in S. cerevisiae and small molecule binding residues across all species to degrees greater than expected by random chance. Metal binding residues are enriched in these entanglements in H. sapiens. Increasing statistical power by pooling together these species data, we find RNA-binding residues are enriched in these entanglement components. On the other hand, there is a spatial depletion of crossing residues at sites involved in protein binding. Using machine learning, we identified eight robust features predictive of these entanglements, achieving AUROC scores of 0.8 across species. These results are significant because they suggest a direct role for components of native entanglements in particular protein functions, as well as identifying strong secondary structure and sequence preferences in native entanglements.

Humans↗
PubMed2026

Mass spectrometry-based mapping of the ubiquitin chaperone code.

Maintenance of proteome integrity is essential for cellular homeostasis and organismal health. This integrity depends on proteostasis, a coordinated network of protein quality control systems that regulate protein folding, stabilization, and degradation. Molecular chaperones, together with proteolytic pathways such as the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway, prevent the accumulation of misfolded and aggregation-prone proteins. Perturbations, including genetic mutations, environmental stress, and aging challenge protein folding fidelity, leading to proteotoxic stress and contributing to the pathogenesis of neurodegenerative disorders. Among the chaperone machinery, the HSP70 and HSP90 families play central roles in maintaining protein conformational homeostasis and directing damaged or misfolded substrates toward refolding or degradation pathways. Recent studies show that chaperone activity is dynamically regulated by diverse post-translational modifications (PTMs), including phosphorylation, acetylation, and ubiquitination, collectively termed the "chaperone code." These modifications modulate chaperone-client interactions, enzymatic activity, localization, and coordination with protein degradation systems. Mass spectrometry (MS)-based proteomics has emerged as a powerful approach for mapping ubiquitination sites and quantifying ubiquitin signaling dynamics. This chapter outlines experimental and computational strategies for MS-based analysis of the ubiquitin chaperone code, including di-glycine peptide enrichment, site identification, quantitative analysis, and validation.

Humans↗