Biochemistry and Pharmacology - Research Publications
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ItemTadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2018-05-11)Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's 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.
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ItemNo Preview AvailableThe Asian Biophysics Association-supporting biophysics in the greater Asia region.(Springer Science and Business Media LLC, 2019-06)
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ItemProtein aggregation in cell biology: An aggregomics perspective of health and disease.(Elsevier, 2020-03)Maintaining protein homeostasis (proteostasis) is essential for cellular health and is governed by a network of quality control machinery comprising over 800 genes. When proteostasis becomes imbalanced, proteins can abnormally aggregate or become mislocalized. Inappropriate protein aggregation and proteostasis imbalance are two of the central pathological features of common neurodegenerative diseases including Alzheimer, Parkinson, Huntington, and motor neuron diseases. How aggregation contributes to the pathogenic mechanisms of disease remains incompletely understood. Here, we integrate some of the key and emerging ideas as to how protein aggregation relates to imbalanced proteostasis with an emphasis on Huntington disease as our area of main expertise. We propose the term "aggregomics" be coined in reference to how aggregation of particular proteins concomitantly influences the spatial organization and protein-protein interactions of the surrounding proteome. Meta-analysis of aggregated interactomes from various published datasets reveals chaperones and RNA-binding proteins are common components across various disease contexts. We conclude with an examination of therapeutic avenues targeting proteostasis mechanisms.
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ItemProbing Protein Solubility Patterns with Proteomics for Insight into Network Dynamics.(Springer US, 2022)Proteome solubility contains latent information on the nature of protein interaction networks in cells and changes in solubility can provide information on rewiring of networks. Here, we report a simple one-step ultracentrifugation method to separate the soluble and insoluble fraction of the proteome. The method involves quantitative proteomics and a bioinformatics strategy to analyze the changes that arise. Because protein solubility changes are also associated with protein misfolding and aggregation in neurodegenerative disease, we also include a protocol for isolating disease-associated protein aggregates with pulse shape analysis (PulSA) by flow cytometry as a complementary approach that can be used alongside the more general measure of solubility or as a stand-alone approach.
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ItemA Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress(AMER CHEMICAL SOC, 2022-05-06)Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.
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ItemNo Preview AvailableWidespread remodeling of proteome solubility in response to different protein homeostasis stresses(National Academy of Sciences, 2020-02-04)The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington’s disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of 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 remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.
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ItemProtein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli(NATURE PORTFOLIO, 2022-11-28)The correct 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. 'Protein painting methods provide a means to address this gap in knowledge by monitoring the conformational status of proteins within cells at the proteome-wide scale. Here, we demonstrate the ability of a protein painting method employing tetraphenylethene maleimide (TPE-MI) to 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.
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ItemArginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate.(Elsevier BV, 2023-01)Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPRs), 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 mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, 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 a source of stress and toxicity and may be a crucial component in pathomechanisms.
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ItemLongitudinal spatial mapping of lipid metabolites reveals pre-symptomatic changes in the hippocampi of Huntington?s disease transgenic mice(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2023-01-01)In Huntington's disease (HD), a key pathological feature includes the development of inclusion-bodies of fragments of the mutant huntingtin protein in the neurons of the striatum and hippocampus. To examine the molecular changes associated with inclusion-body formation, we applied MALDI-mass spectrometry imaging and deuterium pulse labelling to determine lipid levels and synthesis rates in the hippocampus of a transgenic mouse model of HD (R6/1 line). The R6/1 HD mice lacked inclusions in the hippocampus at 6 weeks of age (pre-symptomatic), whereas inclusions were pervasive by 16 weeks of age (symptomatic). Hippocampal subfields (CA1, CA3 and DG), which formed the highest density of inclusion formation in the mouse brain showed a reduction in the relative abundance of neuron-enriched lipids that have roles in neurotransmission, synaptic plasticity, neurogenesis, and ER-stress protection. Lipids involved in the adaptive response to ER stress (phosphatidylinositol, phosphatidic acid, and ganglioside classes) displayed increased rates of synthesis in HD mice relative to WT mice at all the ages examined, including prior to the formation of the inclusion bodies. Our findings, therefore, support a role for ER stress occurring pre-symptomatically and potentially contributing to pathological mechanisms underlying HD.