Browsing by Subject "Proteostasis"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs
    (Elsevier, 2020) Stevens, Mckayla; Howe, Chris; Ray, Anne-Marie; Washburn, Alex; Chitre, Siddhi; Sivinski, Jared; Park, Yangshin; Hoang, Quyen Q.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.
  • Thumbnail Image
    Item
    Analysis of translatomic changes in the Ubqln2P497S model of ALS reveals that motor neurons express muscle-associated genes in non-disease states
    (Frontiers Media, 2024-11-19) Stansberry, Wesley M.; Fiur, Natalie C.; Robins, Melissa M.; Pierchala, Brian A.; Anatomy, Cell Biology and Physiology, School of Medicine
    Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressively worsening motor symptoms that lead to eventual fatal paralysis. The number of gene mutations associated with ALS have increased dramatically in recent years, suggesting heterogeneity in the etiology of ALS and the need to develop new models of the disease that encompass these pathologies. In 2011, mutations in the UBQLN2 gene were identified in families with both ALS and frontotemporal dementia (FTD) and have since been linked to ubiquitinated TDP43 inclusion pathology. The involvement of UBQLN2 in ubiquitination and proteasome function suggests an important role in proteostasis, which is reported to be impaired in ALS. Methods: A UBQLN2 mouse model was generated for the P497S mutation and recapitulates some of the motor symptoms of ALS. We utilized ribosomal profiling followed by mRNA sequencing of associated transcripts to characterize gene expression changes of motor neurons in the Ubqln2P497S model and evaluated ALS phenotypes in these animals. Results: At 12 months of age, we observed reduced motor neuron survival and neuromuscular junction denervation in these mice that translated into motor deficits observed in locomotor behavioral trials. The sequencing of motor neuron transcripts revealed that Wnt pathways and muscle-related transcripts were downregulated in Ubqln2P497S mice, while metabolic pathways were upregulated. Discussion: Surprisingly, genes often reported to be muscle-specific, such as Desmin and Acta1, were expressed in motor neurons and were dramatically downregulated in symptomatic Ubqln2P497S mice. The expression of muscle transcripts by motor neurons suggests their potentially supportive role in skeletal muscle maintenance.
  • Thumbnail Image
    Item
    Bis-aryl-α,β-unsaturated ketone (ABK) chaperonin inhibitors exhibit selective cytotoxicity to colorectal cancer cells that correlates with levels of aberrant HSP60 in the cytosol
    (Elsevier, 2022) Chitre, Siddhi; Ray, Anne-Marie; Stevens, Mckayla; Doud, Emma H.; Liechty, Hope; Washburn, Alex; Tepper, Katelyn; Sivinski, Jared; O’Hagan, Heather M.; Georgiadis, Millie M.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,β-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.
  • Thumbnail Image
    Item
    Combating Parkinson's disease-associated toxicity by modulating proteostasis
    (National Academy of Sciences, 2017-01-31) Park, Yangshin; Hoang, Quyen Q.; Biochemistry and Molecular Biology, School of Medicine
  • Thumbnail Image
    Item
    Does Data-Independent Acquisition Data Contain Hidden Gems? A Case Study Related to Alzheimer's Disease
    (American Chemical Society, 2022) Hubbard, Evan E.; Heil, Lilian R.; Merrihew, Gennifer E.; Chhatwal, Jasmeer P.; Farlow, Martin R.; McLean, Catriona A.; Ghetti, Bernardino; Newell, Kathy L.; Frosch, Matthew P.; Bateman, Randall J.; Larson, Eric B.; Keene, C. Dirk; Perrin, Richard J.; Montine, Thomas J.; MacCoss, Michael J.; Julian, Ryan R.; Pathology and Laboratory Medicine, School of Medicine
    One of the potential benefits of using data-independent acquisition (DIA) proteomics protocols is that information not originally targeted by the study may be present and discovered by subsequent analysis. Herein we reanalyzed DIA data originally recorded for global proteomic analysis to look for isomerized peptides, which occur as a result of spontaneous chemical modifications to long-lived proteins. Examination of a large set of human brain samples revealed a striking relationship between Alzheimer’s disease (AD) status and isomerization of aspartic acid in a peptide from tau. Relative to controls, a surprising increase in isomer abundance was found in both autosomal dominant and sporadic AD samples. To explore potential mechanisms that might account for these observations, quantitative analysis of proteins related to isomerization repair and autophagy was performed. Differences consistent with reduced autophagic flux in AD-related samples relative to controls were found for numerous proteins, including most notably p62, a recognized indicator of autophagic inhibition. These results suggest, but do not conclusively demonstrate, that lower autophagic flux may be strongly associated with loss of function in AD brains. This study illustrates that DIA data may contain unforeseen results of interest, and may be particularly useful for pilot studies investigating new research directions. In this case, a promising target for future investigations into the therapy and prevention of AD has been identified.
  • Thumbnail Image
    Item
    Exploiting the HSP60/10 Chaperonin System as a Chemotherapeutic Target for Colorectal Cancer
    (Elsevier, 2021) Ray, Anne-Marie; Salim, Nilshad; Stevens, Mckayla; Chitre, Siddhi; Abdeen, Sanofar; Washburn, Alex; Sivinski, Jared; O’Hagan, Heather M.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.
  • Thumbnail Image
    Item
    HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules
    (Elsevier, 2019-05-01) Stevens, Mckayla; Abdeen, Sanofar; Salim, Nilshad; Ray, Anne-Marie; Washburn, Alex; Chitre, Siddhi; Sivinski, Jared; Park, Yangshin; Hoang, Quyen Q.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.