Biology subjects

Hatters, D. M.

Publications and source records attributed to Hatters, D. M..

12 recordsLinked to original sources

bioRxiv2025

ASAP-ID: Proximity labelling with small tags

Biotinylation-based proximity labelling methods are valuable for discovering protein-protein interactions within cellular systems. However, one limitation of these approaches is that most require fusing the target protein with the enzyme that biotinylates nearby proteins (i.e., TurboID or APEX2), which risks sterically disrupting the proteins native function. Here, we present a method designed to reduce the steric impact of these fusions and offer greater flexibility in labelling modalities. The method, Antibody and Small-tag Assembly on Proteins for Interaction Detection (ASAP-ID), involves a bipartite system. Target proteins are fused to a small peptide antigen that recruits TurboID or APEX2 fused to an antibody directed to the antigen. Using two different antigen/antibody systems (SunTag and MoonTag), we show that ASAP-ID can specifically label human Lamin A in cells. The method works when the target protein and nanobody are co-expressed together in cis (ASAP-IDIC). We also demonstrate that the approach works when the antibody fusion is added in trans to fixed cells post-expression (ASAP-IDIT). ASAP-IDIT identified more than 448 known and previously undescribed interactors of lamin. We further used ASAP-IDIT to study how ALS-mutant profilin 1 affected its interactome. The method identified proteins involved in protein quality control that correlated with aggregation propensity. Moreover, the different mutants showed variation in the cellular location where aggregates formed. ASAP-IDIT revealed preferences for mitochondrial proteins for the two profilin mutants that tend to aggregate in the cytoplasm, C71G and M114T, and nuclear proteins for a mutant more prone to nuclear aggregation. These findings position ASAP-ID as a powerful addition to the proximity labelling toolkit, capable of probing subtle differences in interactomes in a less invasive manner.

biochemistry↗

bioRxiv2022

Cellular aging impedes stress-activation of a crucial JDP-Hsp70 protein disaggregase

The collapse of protein homeostasis manifests itself in a toxic protein aggregation cascade, which is associated with degenerative diseases and aging. To solubilize aggregates, dedicated protein disaggregases exist in unicellular organisms, but these have no nuclear/cytosolic orthologs in metazoa. Alternative metazoan disaggregation machines have been described, but how these are operated and regulated in vivo remained unknown. We show that protein disaggregases are functionally diversified in human cells to efficiently target different types of stress-induced aggregates in sequential and temporally distinct phases. In particular, we show the selective assembly of an Hsp70-DNAJA1-DNAJB1 trimeric disaggregase that forms during late phase of stress recovery., i.e., after VCP-dependent solubilization of non-native proteins that accumulate in cellular condensates such as nucleoli or stress granules. When activated, the trimeric disaggregase provides resistance to stress toxicity and contributes to amyloid disposal. Strikingly, this disaggregase collapses early in cells undergoing replicative aging with important underlining pathophysiological consequences.

cell biology↗

bioRxiv2022

Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli.

Accurate spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. To address this need, protein painting methods have emerged as a way to gain insight into the conformational status of proteins within cells at the proteome-wide scale. For example, tetraphenylethene maleimide (TPE-MI) has previously been used to quantify the engagement of quality control machinery with client proteins in cell lysates. Here, we showcase the ability of TPE-MI to additionally reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to individual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.

cell biology↗

bioRxiv2022

Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate

Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of DPR-induced stalling and examined whether known regulatory mechanisms of ribosome quality control (RQC) are involved to sense and resolve the stalls. We demonstrate that arginine-containing DPRs lead to stalling in a length dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40xGly-Xxx DPRs shows that stalling is most pronounced where Xxx are positively charged amino acids (Arg or Lys). Through a genome-wide knockout screen we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, respond differently to the readthrough of arginine-rich DPRs. Indeed, we find evidence that DPR-mediated stalling has no natural regulatory responses even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as posing a particular danger to cellular health and viability.

biochemistry↗

bioRxiv2021

Sequence grammar underlying unfolding and phase separation of globular proteins

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs. HighlightsO_LIUnfolded states of globular proteins phase separate to form UPODs in cells C_LIO_LIThe fraction of unfolded molecules and the sticker grammar govern phase separation C_LIO_LIHydrophobic residues act as stickers that engage in intermolecular interactions C_LIO_LISticker grammar also influences gain-of-function recruitment into aberrant UPODs C_LI

biophysics↗

bioRxiv2021

A biosensor to gauge protein homeostasis resilience differences in the nucleus compared to cytosol of mammalian cells

An extensive network of chaperones and other proteins maintain protein homeostasis and guard against inappropriate protein aggregation that is a hallmark of neurodegenerative diseases. Using a fluorescence resonance energy-based biosensor that simultaneously reports on intact cellular chaperone holdase activity and detrimental aggregation propensity, we investigated the buffering capacity of the systems managing protein homeostasis in the nucleus of the human cell line HEK293 compared to the cytosol. We found that the nucleus showed lower net holdase activity and reduced capacity to suppress protein aggregation, suggesting that the nuclear quality control resources are less effective compared to those in the cytosol. Aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to deplete cytosolic chaperone supply by depleting holdase activity. Unexpectedly, the same stress increased holdase activity in the nucleus suggesting that proteostasis stress can trigger a rebalance of chaperone supply in different subcellular compartments. Collectively the findings suggest the cytosol has more capacity to manage imbalances in proteome foldedness than the nucleus, but chaperone supply can be redirected into the nucleus under conditions of proteostasis stress caused by cytosolic protein aggregation.

biochemistry↗

bioRxiv2021

Hidden information on protein function in censuses of proteome foldedness

Methods that assay protein foldedness with proteomics have generated censuses of protein folding stabilities in biological milieu. Surprisingly, different censuses poorly correlate with each other. Here, we show that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding. About one quarter of cysteine or methionine sidechains in proteins in mammalian cell lysate increase in reactivity upon chemical denaturant titration consistent with two-state unfolding. Paradoxically, up to one third decreased reactivity, which were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings. One Sentence SummaryWe show that proteome folding stability censuses are ill-defined because they earmark hidden information on conformation and ligand binding.

biochemistry↗

bioRxiv2020

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

Mutations that cause Huntingtons Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntingtons disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and examined whether ribosome quality control machinery previously implicated in stalled ribosomes were involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation.

cell biology↗

bioRxiv2020

Inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein degradation

Poly(glycine-alanine) (polyGA) is one of the dipolypeptides expressed in Motor Neuron Disease caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ) in Huntingtons disease and both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms to confer cellular dysfunction similarly. Here we investigated which endogenous proteins were enriched in these inclusions and whether aggregation-prone lengths of polyQ (Q97), in context of Huntingtin exon 1, shared similar patterns to aggregation-prone lengths of polyGA (101GA). When co-expressed in the same cell, polyGA101 and HttQ97 inclusions adopted distinct phases with no overlap suggesting different endogenous proteins would be enriched. Proteomic analyses indeed yielded distinct sets of endogenous proteins recruited into the inclusion types. The proteosome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in polyQ aggregates. Both structures revealed a synergy of degradation machinery including proteins in the polyQ aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.

biochemistry↗

bioRxiv2019

Arginine valency in C9ORF72 dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a Neuro2a cell model to demonstrate that the valency of Arg in the most toxic DPRS, PR and GR, drives promiscuous binding to the proteome, compared to a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats robustly stalled the ribosome suggesting high-valency Arg electrostatically jams the ribosome exit tunnel during synthesis. Poly-GR also bound to arginine methylases and induced hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.\n\nSIGNIFICANCE STATEMENTThe major genetic cause of MND are mutations in an intron of the C9ORF72 gene that lead to the expansion in the length of a hexanucleotide repeat sequence, and subsequent non-AUG mediated translation of the intron into 5 different DPRs. The two DPRs containing Arg are potently toxic in animal and cell models. Our research shows that the valency of Arg mediates widespread proteome binding especially affecting machinery involved in Arg-methylation, cytoskeleton and translation. We suggest the mechanisms for toxicity are multipronged and involve electrostatic jamming of ribosomes during translation, acting as substrate mimetics for arginine methylase activity that renders the endogenous proteome hypomethylated and impairing actin cytoskeleton assembly. These mechanisms explain pathologic signatures previous reported in human brain pathology.

biochemistry↗

bioRxiv2019

Widespread remodelling of proteome solubility in response to different protein homeostasis stresses

The accumulation of protein deposits in neurodegenerative diseases involves the presence of a metastable subproteome vulnerable to aggregation. To investigate this subproteome and the mechanisms that regulates it, we measured the proteome solubility of the Neuro2a cell line under protein homeostasis stresses induced by Huntington Disease proteotoxicity; Hsp70, Hsp90, proteasome and ER-mediated folding inhibition; and oxidative stress. We found one-quarter of the proteome extensively changed solubility. Remarkably, almost all the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodelling of protein complexes involved in adaptation to perturbation, most notably stress granule proteins, which responded differently to different stresses. These results indicate that the robustness of protein homeostasis relies on the absence of proteins highly vulnerable to aggregation and on large changes in aggregation state of regulatory mechanisms that restore protein solubility upon specific perturbations.

biochemistry↗

bioRxiv2017

Tadpole-like conformations of huntingtin exon 1 with expanded polyglutamine engenders novel interactions in cells

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntingtons disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

biochemistry↗