Biology subjects

Balch, W. E.

Publications and source records attributed to Balch, W. E..

11 recordsLinked to original sources

bioRxiv2025

Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity

Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically-diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a compound that promotes adaptive ER proteostasis remodeling through mechanisms including preferential activation of the ATF6 signaling arm of the unfolded protein response (UPR). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a potential molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated 1-antitrypsin (A1AT) variant and an epilepsy-associated GABAA receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically-diverse protein misfolding disorders.

cell biology↗

bioRxiv2022

Profiling Genetic Diversity Reveals the Molecular Basis for Balancing Function with Misfolding in Alpha-1 Antitrypsin

Genetic variation of alpha-1 antitrypsin (AAT) is responsible for alpha-1-antitrypsin deficiency (AATD) leading to gain-of-toxic aggregation in the liver and loss-of-function on neutrophil elastase (NE) inhibitory activity in the lung contributing to chronic obstructive pulmonary disease (COPD) during aging. To probe the molecular basis for how biology designs the protein fold to achieve balance between sequence, function and structure contributing to AATD in the population, we measured the intracellular monomer and polymer, secreted monomer and polymer and NE inhibitory activity of 75 alpha-1-antitrypsin (AAT) variants. To address the complex folding dynamics affecting the form and function of the protein fold that is differentially impacted by variants in the population, we applied a Gaussian process regression (GPR) based machine learning approach termed variation spatial profiling (VSP). By using a sparse collection of extant variants to link genotype to phenotype, VSP maps spatial covariance (SCV) relationships that quantitate the functional value of every residue in the wild-type (WT) AAT sequence with defined uncertainty in the context of its protein fold design. The SCV-based uncertainty allows us to pinpoint critical short- and long-range residue interactions involving 3 regions-the N-terminal (N1), middle (M2) and carboxyl-terminal (C3) of AAT polypeptide sequence that differentially contribute to the balance between function and misfolding of AAT, thus providing an unanticipated platform for precision therapeutic development for liver and lung disease. By understanding mechanistically the complex fold design of the metastable WT AAT fold, we posit that GPR-based SCV provides a foundation for understanding the evolutionary design of the fold from the ensemble of structures found in the population driving biology for precision management of AATD in the individual.

molecular biology↗

bioRxiv2022

Covariant Fitness Clusters Reveal Structural Evolution of SARS-CoV-2 Polymerase Across the Human Population

Understanding the fitness landscape of viral mutations is crucial for uncovering the evolutionary mechanisms contributing to pandemic behavior. Here, we apply a Gaussian process regression (GPR) based machine learning approach that generates spatial covariance (SCV) relationships to construct stability fitness landscapes for the RNA-dependent RNA polymerase (RdRp) of SARS- CoV-2. GPR generated fitness scores capture on a residue-by-residue basis a covariant fitness cluster centered at the C487-H642-C645-C646 Zn2+ binding motif that iteratively evolves since the early phase pandemic. In the Alpha and Delta variant of concern (VOC), multi-residue SCV interactions in the NiRAN domain form a second fitness cluster contributing to spread. Strikingly, a novel third fitness cluster harboring a Delta VOC basal mutation G671S augments RdRp structural plasticity to potentially promote rapid spread through viral load. GPR principled SCV provides a generalizable tool to mechanistically understand evolution of viral genomes at atomic resolution contributing to fitness at the pathogen-host interface.

evolutionary biology↗

bioRxiv2019

Proteostasis and Energetics as Proteome Hallmarks of Aging and Influenza Challenge in Pulmonary Disease

Aging is associated with an increased risk for the development of many diseases. This is exemplified by the increased incidence of lung injury, muscle dysfunction and cognitive impairment in the elderly following influenza infection. Because the infectious cycle of flu is dependent upon the properties of the host, we examined the proteome of alveolar macrophages (AM) and type 2 cells (AT2) obtained from young (3 months) and old (18 months) naive mice and mice exposed to influenza A. Our proteomics data show that there is a maladaptive collapse of the proteostasis network (PN) and changes in mitochondrial pathways in the aged naive AM and AT2 proteomes. The mitochondrial imbalance and proteostatic collapse seen in aged cells places an excessive folding burden on these cells, which is further exacerbated following exposure to influenza A. Specifically, we see an imbalance in Hsp70 co-chaperones involved in protein folding and Hsp90 co-chaperones important for stress signaling pathways that are essential for cellular protection during aging. The acute challenge of influenza A infection of young and aged AM and AT2 cells reveals that age-associated changes in the chaperome affect the ability of these cells to properly manage the infection and post-infection biology, contributing to cytotoxicity. We posit that proteomic profiling of individual cell type specific responses provides a high impact approach to pinpoint fundamental molecular relationships that may contribute to the susceptibility to aging and environmental stress, providing a platform to identify new targets for therapeutic intervention to improve resiliency in the elderly.

cell biology↗

bioRxiv2019

Correction of Niemann-Pick type C1 disease with the histone deacetylase inhibitor valproic acid

Niemann-Pick type C (NPC) disease is primarily caused by mutations in the NPC1 gene and is characterized by the accumulation of unesterified cholesterol and lipids in the late endosomal (LE) and lysosomal (Ly) compartments. The most prevalent disease-linked mutation is the I1061T variant of NPC1, which exhibits defective folding and trafficking from the endoplasmic reticulum to the LE/Ly compartments. We now show that the FDA-approved histone deacetylase inhibitor (HDACi) valproic acid (VPA) corrects the folding and trafficking defect associated with I1061T-NPC1 leading to restoration of cholesterol homeostasis, an effect that is largely driven by a reduction in HDAC7 expression. The VPA-mediated trafficking correction is in part associated with an increase in the acetylation of lysine residues in the cysteine-rich domain of NPC1. The HDACi-mediated correction is synergistically improved by combining it with the FDA-approved anti-malarial, chloroquine, a known lysosomotropic compound, which improved the stability of the LE/Ly-localized fraction of the I1061T variant. We posit that combining the activity of VPA, to modulate epigentically the cellular acetylome, with chloroquine, to alter the lysosomal environment to favor stability of the trafficked I1061T variant protein, can have a significant therapeutic benefit in patients carrying at least one copy of the I1061T variant of NPC1, the most common disease-associated mutation leading to NPC disease. Given its ability to cross the blood brain barrier, we posit VPA provides a potential mechanism to improve the response to 2-hydroxypropyl-{beta}-cyclodextrin, by restoring functional NPC1 to cholesterol managing compartment as an adjunct therapy.

cell biology↗

bioRxiv2019

The Aging Microenvironment Shapes Alveolar Macrophage Identity in Aging

A dysfunctional response to inhaled pathogens and toxins drives a substantial portion of the susceptibility to acute and chronic lung disease in the elderly. We used transcriptomic profiling combined with genetic lineage tracing, heterochronic adoptive transfer, parabiosis and treatment with metformin to show that the lung microenvironment defines the phenotype of long-lived alveolar macrophages during aging. While tissue-resident alveolar macrophages persist in the lung without input from monocytes over the lifespan, severe lung injury results in their replacement with monocyte-derived alveolar macrophages. These monocyte-derived alveolar macrophages are also shaped by the microenvironment both during aging and in response to a subsequent environmental challenge to become transcriptionally and functionally similar to tissue-resident alveolar macrophages. These findings show that changes in alveolar macrophage phenotypes during injury and aging are not cell autonomous but instead are shaped by changes in the aging lung microenvironment.

immunology↗

bioRxiv2019

A reference map of the human proinsulin biosynthetic interaction network

The beta-cell protein synthetic machinery is dedicated to the production of insulin, which plays a critical role in organismal homeostasis. Insulin synthesis requires the proper folding and trafficking of its precursor, proinsulin, yet the precise network of proinsulin protein interactions in the secretory pathway remains poorly defined. In the present study we conducted unbiased profiling of the proinsulin interactome in human islets, utilizing a human proinsulin-specific monoclonal antibody for affinity purification and mass spectrometry. Stringent analysis identified a central node of interactions between human proinsulin and sequential secretory pathway proteins that is remarkably conserved across 3 ethnicities and both genders. Among the most prominent proinsulin interactions was with ER-localized peroxiredoxin-4 (PRDX4). A functional role for PRDX4 in beta-cells was demonstrated by gene silencing that rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress. Conversely, exogenous PRDX4 improved proinsulin folding. Notably, oxidative stress and even high glucose treatment alone induced proinsulin misfolding in human islets and MIN6 cells, and this was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. This finding prompted PRDX4 analysis in a panel of human islet samples that revealed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of healthy individuals. Taken together, these data highlight the importance of elucidating the complete proinsulin interactome in human islets in order to understand critical steps controlling insulin biosynthesis, beta cell function, and T2D.

biochemistry↗

bioRxiv2019

Aging is associated with a systemic length-driven transcriptome imbalance

Aging manifests itself through a decline in organismal homeostasis and a multitude of cellular and physiological functions1. Efforts to identify a common basis for vertebrate aging face many challenges; for example, while there have been documented changes in the expression of many hundreds of mRNAs, the results across tissues and species have been inconsistent2. We therefore analyzed age-resolved transcriptomic data from 17 mouse organs and 51 human organs using unsupervised machine learning3-5 to identify the architectural and regulatory characteristics most informative on the differential expression of genes with age. We report a hitherto unknown phenomenon, a systemic age-dependent length-driven transcriptome imbalance that for older organisms disrupts the homeostatic balance between short and long transcript molecules for mice, rats, killifishes, and humans. We also demonstrate that in a mouse model of healthy aging, length-driven transcriptome imbalance correlates with changes in expression of splicing factor proline and glutamine rich (Sfpq), which regulates transcriptional elongation according to gene length6. Furthermore, we demonstrate that length-driven transcriptome imbalance can be triggered by environmental hazards and pathogens. Our findings reinforce the picture of aging as a systemic homeostasis breakdown and suggest a promising explanation for why diverse insults affect multiple age-dependent phenotypes in a similar manner.

systems biology↗

bioRxiv2018

Managing the Spatial Covariance of Genetic Diversity in Niemann-Pick C1 Through Modulation of the Hsp70 Chaperone System

Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold to allow biology to evolve through variation in the population and in response to the environment. Failure in proteostasis can trigger multiple disease states affecting both human health and lifespan. Niemann-Pick C (NPC) disease is a genetic disorder mainly caused by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes and lysosomes (LE/Ly) to manage cholesterol homeostasis. Proteostatic defects triggered by >600 NPC1 variants found in the human population inhibit export of NPC1 protein from ER or function in downstream LE/Ly, leading to accumulation of cholesterol and rapid onset neurodegeneration in childhood for most patients. We now show that chemical allosteric inhibitors, such as JG98, targeting the cytosolic Hsp70 chaperone/co-chaperone complex improves the trafficking and stability of NPC1 variants with diverse NPC1 genotypes. By exploiting the knowledge-base of NPC1 variants found in the world-wide patient population using Variation Spatial Profiling (VSP), a Gaussian-process based machine learning (ML) approach, we show how the Hsp70 chaperone system alters the spatial covariance (SCV) tolerance of the ER and the SCV set-points for each residue of the NPC1 polypeptide chain differentially to improve trafficking efficiency and post-ER stability for variants distributed across the entire NPC1 polypeptide. The impact of JG98 is supported by the observation that silencing of Hsp70 specific nucleotide exchange factors (NEF) (BCL-anthogene (BAG) family) co-chaperones significantly improve the folding status of NPC1 variants. Together, these studies suggest that targeting the cytosolic Hsp70 system to adjust the SCV tolerance of the proteostasis network can improve recognition of the plasticity of the NPC1 fold found in the disease population for trafficking to the LE/Ly compartments.

genomics↗

bioRxiv2018

Management of Hsp90-Dependent Protein Folding by Small Molecule Targeting the Aha1 Co-Chaperone

The core cytosolic Hsp90 chaperone/co-chaperone complex plays a critical role in proteostasis management of human health and disease. To identify novel compounds that alter the ability of the Hsp90 co-chaperone Aha1 to modulate the ATPase activity found in multiple folding diseases ranging from steroid hormone receptor (SHR) sensitive prostate cancer to tauopathies associated with neurodegenerative diseases, we employed a high throughput screening (HTS) assay to monitor selectively Aha1-stimulated Hsp90 (ASH) ATPase activity. The ASH assay identified SEW04784 (SEW), a small molecule that disrupts ASH activity without inhibiting the basal Hsp90 ATPase activity. NMR analysis reveals that SEW binds to the C-terminal domain of Aha1 to disrupt its asymmetric binding to Hsp90 leading to abrogation of its chaperoning activity of Hsp90. SEW exhibits therapeutic potential by blocking the transcriptional activity of prostate cancer (PCa) associated variants of the androgen receptor (AR) in a cell-based model of PCa. Additionally, SEW exhibits the ability to clear toxic, phosphorylated tau aggregated species associated with tauopathies. By not directly impacting the basal ATPase function of the abundant and ubiquitous Hsp90, SEW could provide a therapeutic approach for mitigation of client-specific proteostatic disease.

biochemistry↗

bioRxiv2018

HDAC Inhibitors Rescue Multiple Disease-Causing CFTR Variants

Understanding the role of the epigenome in protein misfolding diseases remains a challenge in light of genetic diversity found in the world-wide population revealed by human genome sequencing efforts and the highly variable respond of the disease population to any therapeutic. An ever-growing body of evidence has shown that histone deacetylase (HDAC) inhibitors (HDACi) can have significant benefit in correcting protein misfolding diseases that occur in response to both familial and somatic mutation. Cystic fibrosis (CF) is a familial autosomal recessive disease, caused by genetic diversity in the CF transmembrane conductance regulator (CFTR) gene, a cAMP-dependent chloride channel expressed at the apical plasma membrane of epithelial cells in multiple tissues. The potential utility of HDACi in correcting F508del as well as the over 2000 CF-associated variants remains controversial. To address this concern, we examined the impact of FDA-approved HDACi on the trafficking and function of a panel of CFTR variants. Our data reveal that panobinostat (LBH-589) and romidepsin (FK-228) provide functional correction of class II and III CFTR variants, restoring cell surface chloride channel activity in primary human bronchial epithelial (hBE) cells. We further demonstrate a synergistic effect of these HDACi with Vx809, that together can significantly restore channel activity for multiple CFTR variants. These data suggest that HDACi can serve to level the cellular playing field for correcting CF-causing mutations, a leveling effect that might also extend to other protein misfolding diseases.

cell biology↗