Biology subjects

Frydman, J.

Publications and source records attributed to Frydman, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Ubiquitin selective ribosome profiling reveals systematic principles of co-translational quality control

Protein biogenesis is a stress- and error-sensitive process that can lead to nascent protein misfolding and aggregation, challenging cellular proteostasis.1 Co-translational ubiquitination (CTU) is a critical surveillance mechanism,2,3 yet its regulatory principles remain unclear due to limited known substrates and the lack of translatome-wide methods to query CTU. Here, we introduce UbSeRP, an approach that enables ubiquitin linkage-specific, and translatome-wide mapping of CTU. Focusing on ribosome-associated quality control (RQC), we expand the known endogenous RQC substrates in S. cerevisiae from few4-7 to thousands, reveal that only a subset of all identified disomes undergoes RQC, and uncover biophysical features of nascent chains that predict RQC engagement. We also identify widespread RQC-independent ubiquitination of nascent proteins, implicating broader roles of CTU in protein complex assembly. In a chronological aging model, we show that aging remodels translation and diminishes RQC engagement, favoring RQC-independent proteasomal pathways. Our findings provide systematic insight into the determinants and adaptability of CTU in maintaining proteostasis under changing physiological conditions.

biochemistry↗

bioRxiv2026

Q-DOAS: A Proximity Quenching Assay for Real-Time Detection of Early Protein Aggregation Events

Accurate measurement of protein aggregation is essential for studying neurodegenerative diseases. The standard ThT assay reports on amyloid formation but is blind to early oligomers and is prone to interference. We describe Q-DOAS, a plate-reader assay that quantifies protein self-assembly in real time via proximity-quenching of a single, site-specifically conjugated dye (BODIPY-TMR). Using mutant Huntingtin-exon 1 (mHTT-Ex1) and -Synuclein A53T, we show Q-DOAS detects pre-amyloid oligomers, yielding quantitative kinetic data compatible with mechanistic analysis. We demonstrate its utility to dissect mutational effects, screen for protein and small-molecule inhibitors, and quantify amyloid seeding activity in cellular and mouse models of Huntingtons disease. Q-DOAS also detects seeds in cerebrospinal fluid from Parkinsons disease patients without amplification. Q-DOAS provides a sensitive, robust, and scalable tool for studying the earliest events in amyloid pathologies and for advancing therapeutic development.

biochemistry↗

bioRxiv2025

NAC controls nascent chain fate through tunnel sensing and chaperone action

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-bound factor with essential yet incompletely understood roles in protein biogenesis. Here, we show that NAC is a multifaceted regulator that coordinates translation elongation, cotranslational folding, and organelle targeting through distinct interactions with nascent polypeptides both inside and outside the ribosome exit tunnel. Using NAC-selective ribosome profiling in C. elegans, we identify thousands of sequence-specific NAC binding events across the nascent proteome, revealing broad cotranslational engagement with hydrophobic and helical motifs in cytosolic, nuclear, ER, and mitochondrial proteins. Unexpectedly, we discover an intra-tunnel sensing mode, where NAC engages ribosomes with extremely short nascent polypeptides inside the exit tunnel in a sequence-specific manner. These early NAC interactions induce an early elongation slowdown that tunes ribosome flux and prevent ribosome collisions, linking NACs chaperone activity to kinetic control of translation. We propose that NAC action protects aggregation-prone intermediates by shielding amphipathic helices thus promoting cytonuclear folding and supporting mitochondrial membrane protein biogenesis and ER targeting by early recognition of signal sequences and transmembrane domain. Our findings establish NAC as an early-acting, multifaceted orchestrator of cotranslational proteostasis, with distinct mechanisms of action on nascent chains depending on their sequence features and subcellular destinations.

biochemistry↗

bioRxiv2024

TRiC/CCT Chaperonin Governs RNA Polymerase II Activity in the Nucleus to Support RNA Homeostasis

The chaperonin TRiC/CCT is a large hetero-oligomeric ringed-structure that is essential in eukaryotes. While present in the nucleus, TRiC/CCT is typically considered to function in the cytosol where it mediates nascent polypeptide folding and the assembly/disassembly of protein complexes. Here, we investigated the nuclear role of TRiC/CCT. Inactivation of TRiC/CCT resulted in a significant increase in the production of nascent RNA leading to the accumulation of noncoding transcripts. The influence on transcription was not due to cytoplasmic TRiC/CCT-activities or other nuclear proteins as the effect was observed when TRiC/CCT was evicted from the nucleus and restricted to the cytoplasm. Rather, our data support a direct role of TRiC/CCT in regulating RNA polymerase II activity, as the chaperonin modulated nascent RNA production both in vivo and in vitro. Overall, our studies reveal a new avenue by which TRiC/CCT contributes to cell homeostasis by regulating the activity of nuclear RNA polymerase II.

molecular biology↗

bioRxiv2023

A comprehensive atlas of the aging vertebrate brain reveals signatures of progressive proteostasis dysfunction

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brains biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules. One-Sentence SummaryTranslation pausing reshapes the aging brain proteome, revealing vulnerabilities in the biogenesis of nucleic-acid protein.

systems biology↗

bioRxiv2023

SARS-CoV-2 Nsp1 regulates translation start site fidelity to promote infection

A better mechanistic understanding of virus-host interactions can help reveal vulnerabilities and identify opportunities for therapeutic interventions. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in polysome composition and protein synthesis during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones whose inhibition impairs infectious particle production without major toxicity to the host. We find that CoV2 non-structural protein Nsp1 selectively enhances virus translation through functional interactions with initiation factor EIF1A. When EIF1A is depleted, more ribosomes initiate translation from an upstream CUG start codon, inhibiting translation of non-structural genes and reducing viral titers. Together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and identifies druggable targets for potential antiviral development.

microbiology↗

bioRxiv2023

Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons

Aging is the most prominent risk factor for Alzheimers disease (AD). However, the cellular mechanisms linking neuronal proteostasis decline to the characteristic aberrant protein deposits in AD brains remain elusive. Here, we develop transdifferentiated neurons (tNeurons) from human dermal fibroblasts as a neuronal model that retains aging hallmarks and exhibits AD-linked vulnerabilities. Remarkably, AD tNeurons accumulate proteotoxic deposits, including phospho-Tau and A{beta}, resembling those in AD patient and APP mouse brains. Quantitative tNeuron proteomics identify aging and AD-linked deficits in proteostasis and organelle homeostasis, most notably in endosome-lysosomal components. Lysosomal deficits in aged tNeurons, including constitutive lysosomal damage and ESCRT-mediated lysosomal repair defects, are exacerbated in AD tNeurons and linked to inflammatory cytokine secretion and cell death. Supporting lysosomal deficits centrality in AD, compounds ameliorating lysosomal function reduce A{beta} deposits and cytokine secretion. Thus, the tNeuron model system reveals impaired lysosomal homeostasis as an early event of aging and AD.

cell biology↗

bioRxiv2023

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chambers equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding. Highlights- Structural analysis of TRiC-mediated folding cycle with cochaperones PhLP2A and PFD. - The interactions of PhLP2A and PFD with TRiC are mutually exclusive. - PhLP2A domains interact in a subunit-specific manner with the TRiC chamber. - PhLP2A domains are rearranged in ATP-closed TRiC to contact actin across the ring interface

biophysics↗

bioRxiv2023

A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

bioinformatics↗

bioRxiv2022

An ESCRT-dependent pathway of Nuclear and Cytoplasmic Spatial PQC is coordinated at Nuclear Vacuolar Junctions

Effective Protein Quality Control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we elucidate the coordination of nuclear and cytoplasmic spatial PQC. While cytoplasmic misfolded proteins concentrate in a cytoplasmic, perinuclear Juxta Nuclear Quality control compartment (JUNQ), nuclear misfolded proteins sequester into a perinucleolar IntraNuclear Quality control compartment (INQ). Particle tracking reveals the INQ and JUNQ converge to face each other across the nuclear envelope at a site proximal to the Nuclear-Vacuolar Junction (NVJ) marked by perinuclear ESCRT-II/-III protein Chm7. Strikingly, this ESCRT-dependent convergence facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. We propose perinuclear ESCRT coordinates spatial PQC at nuclear-vacuolar contacts to facilitate vacuolar clearance of nuclear and cytoplasmic misfolded proteins.

biochemistry↗

bioRxiv2022

BDNF and TRiC-inspired Reagents Rescue Cortical Synaptic Deficits in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) results from a CAG repeat expansion in the gene for Huntington (HTT) resulting in expansion of the polyglutamine (Q) tract in the mutant protein (mHTT). Synaptic changes are early manifestations of neuronal dysfunction in HD. However, the mechanism(s) by which mHTT impacts synapse formation and function is not well defined. Herein we explored HD pathogenesis in the BACHD and the {Delta}N17-BACHD mouse models of HD by examining cortical synapse formation and function in primary cultures maintained for up to 35 days (DIV35). We identified synapses by immunostaining with antibodies against pre-synaptic (Synapsin 1) and a post-synaptic (PSD95) marker. Consistent with earlier studies, cortical neurons from both WT and the HD models began to form synapses at DIV14; at this age there were no genotypic differences in synapse numbers. However, from DIV21 through DIV35 BACHD neurons showed progressively smaller numbers of synapses relative to WT neurons. Remarkably, BACHD synaptic deficits were completely rescued by treating cultures with BDNF. Building on earlier studies using reagents inspired by the chaperonin TRiC, we found that addition of the recombinant apical domain of CCT1 partially rescued synapse number. Unexpectedly, unlike BACHD cultures, synapses in {Delta}N17-BACHD cultures showed a progressive increase in number as compared to WT neurons, thus distinguishing synaptic changes in these HD models. Using multielectrode arrays, we discovered age-related functional deficits in BACHD cortical cultures with significant differences present by DIV28. As for synapse number, BDNF treatment prevented most synaptic deficits, including mean firing rate, spikes per burst, inter-burst interval, and synchrony. The apical domain of CCT1 showed similar, albeit less potent effects. These data are evidence that deficits in HD synapse number and function can be replicated in vitro and that treatment with either BDNF or a TRiC-inspired reagent can prevent them. Our findings support the use of cellular models to further explicate HD pathogenesis and its treatments.

neuroscience↗

bioRxiv2022

SARS-CoV2 associated secretion of nanoLuciferase reports on virus and Virus-Like Particle production

SARS-CoV2 is a positive-strand RNA virus in the Coronaviridae family that has caused world-wide morbidity and mortality. While much progress has been made we still need expanded rapid anti-virals. The top advanced antiviral candidates all target stages of RNA replication, leaving virus assembly an unexplored avenue of antiviral research. To address this gap, and explore the biochemical and cell biological features of viral assembly, we have employed an improved virus-like particle (VLP) system. We exploited the small nanoLuciferase protein for enhanced signal and surprisingly found that the protein itself appears to be packaged into both SARS CoV2 VLPs and virions and secreted from cells. Interestingly, nLuc is not co-secreted with dengue or Zika infection, suggesting the large virion of Coronavirus can encaspidate and secrete a cellularly expressed reporter protein. Our findings open the way for powerful new approaches to measure viral particle production, egress and viral entry mechanisms.

molecular biology↗

bioRxiv2022

mTOR inhibitor-independent Autophagy Activator Ameliorates Cellular Tauopathy and Prionopathy Neurodegeneration Phenotypes

Autophagy-lysosomal impairment is an early and prominent feature of neurodegeneration. Autophagy activation reduces protein aggregates and lipid level abnormalities. We performed a high-content imaging-based screen assessing 940,000 small molecules to identify those that reduce lipid droplet numbers. Of 77 validated, structurally diverse hits, 24 increased autophagy flux reporter activity, consistent with accelerated lipid droplet clearance by lipophagy. Of these, we show that CCT020312 activates autophagy independently of mammalian target of rapamycin (mTOR) inhibition, to avoid immunosuppression. CCT020312 reduced insoluble phosphorylated tau levels and tau-mediated neuronal stress vulnerability, as well as reducing intracellular A{beta} levels within directly induced neurons bearing epigenetic marks of aging derived from Alzheimers patient fibroblasts. Moreover, CCT020312 cleared mutant prion protein aggregates and normalized trafficking deficiencies in axons of a cellular model of familial prion disease. Autophagy is widely considered a promising strategy to attenuate neurodegeneration, and here we introduce a strategy to discover new pharmacology.

cell biology↗

bioRxiv2022

A Comprehensive Enumeration of the Human Proteostasis Network. 1. Components of Translation, Protein Folding, and Organelle-Specific Systems

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,500 genes that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology with direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. Here, we provide a curated list of 959 unique genes that comprise the protein synthesis machinery, chaperones, folding enzymes, systems for trafficking proteins into and out of organelles, and organelle-specific degradation systems. In subsequent manuscripts, we will delineate the human autophagy-lysosome pathway, the ubiquitin-proteasome system, and the proteostasis networks of model organisms.

bioinformatics↗

bioRxiv2022

Cancers adapt to their mutational load by buffering protein misfolding stress

In asexual populations that dont undergo recombination, such as cancer, deleterious mutations are expected to accrue readily due to genome-wide linkage between mutations. Despite this mutational load of often thousands of deleterious mutations, many tumors thrive. How tumors survive the damaging consequences of this mutational load is not well understood. Here, we investigate the functional consequences of mutational load in 10,295 human tumors by quantifying their phenotypic response through changes in gene expression. Using a generalized linear mixed model (GLMM), we find that high mutational load tumors up-regulate proteostasis machinery related to the mitigation and prevention of protein misfolding. We replicate these expression responses in cancer cell lines and show that the viability in high mutational load cancer cells is strongly dependent on complexes that degrade and refold proteins. This indicates that upregulation of proteostasis machinery is causally important for high mutational burden tumors and uncovers new therapeutic vulnerabilities. Statement of SignificanceCancers can successfully survive an accumulation of thousands of protein-damaging mutations. Here, we show that high mutational load tumors mitigate these damaging consequences by up-regulating complexes that buffer against protein misfolding stress - providing novel therapeutic vulnerabilities and suggesting that disruption of proteostasis is a hallmark of somatic evolution.

cancer biology↗

bioRxiv2022

Ribotoxic collisions on CAG expansions disrupt proteostasis and stress responses in Huntington's Disease

Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by CAG trinucleotide repeat expansions encoding a polyglutamine (polyQ) tract in the Huntingtin (HTT) gene1. Although mutant HTT (mHTT) protein tends to aggregate, the exact causes of neurotoxicity in HD remain unclear2. Here we show that altered elongation kinetics on CAG expansions cause ribosome collisions that trigger ribotoxicity, proteotoxicity and maladaptive stress responses. CAG expansions cause an elongation rate conflict during HTT translation, when ribosomes rapidly decoding the optimal polyQ encounter a flanking slowly-decoded polyproline tract. The ensuing ribosome collisions lead to premature termination and release of aggregation-prone mHTT fragments. Due to the presence of a stress-responsive upstream open reading frame (uORF), HTT translation and aggregation are limited under normal conditions but enhanced under stress, seeding a vicious cycle of dysfunction. mHTT further exacerbates ribotoxicity by progressively sequestering eIF5A, a key regulator of translation elongation, polyamine metabolism and stress responses. eIF5A depletion in HD cells leads to widespread ribosome pausing on eIF5A-dependent sites, impaired cotranslational proteostasis, disrupted polyamine metabolism and maladaptive stress responses. Importantly, drugs that reduce translation initiation attenuate ribosome collisions and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

neuroscience↗

bioRxiv2022

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

Huntingtons Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics to show differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

neuroscience↗

bioRxiv2022

Structural visualization of the tubulin folding pathway directed by eukaryotic chaperonin TRiC

SUMMARYThe ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of {beta}-tubulin using human Prefoldin and TRiC. We find unstructured {beta}-tubulin is delivered by Prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. CryoEM resolves four near-atomic resolution structures containing progressively folded {beta}-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N-domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Disordered CCT C-termini within the chamber promote subsequent folding of tubulin Core and Middle domains and GTP-binding. Thus, TRiCs chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

biochemistry↗