IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied.

Phage-layer interferometry: a companion diagnostic for phage therapy and a bacterial testing platform.

The continuing and rapid emergence of antibiotic resistance (AMR) calls for innovations in antimicrobial therapies. A promising, 're-emerging' approach is the application of bacteriophage viruses to selectively infect and kill pathogenic bacteria, referred to as phage therapy. In practice, phage therapy is personalized and requires companion diagnostics to identify efficacious phages, which are then formulated into a therapeutic cocktail.

SLC26A9 is selected for endoplasmic reticulum associated degradation (ERAD) via Hsp70-dependent targeting of the soluble STAS domain.

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis.

Heat Shock Protein 70 as a Sex-Skewed Regulator of α-Synucleinopathy.

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein inclusions more robustly in male-derived neurons.

RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.

TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control.

Chaperoning Endoplasmic Reticulum-Associated Degradation (ERAD) and Protein Conformational Diseases.

Misfolded proteins compromise cellular homeostasis. This is especially problematic in the endoplasmic reticulum (ER), which is a high-capacity protein-folding compartment and whose function requires stringent protein quality-control systems. Multiprotein complexes in the ER are able to identify, remove, ubiquitinate, and deliver misfolded proteins to the 26S proteasome for degradation in the cytosol, and these events are collectively termed ER-associated degradation, or ERAD.

Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding.

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson's Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K inward rectifying channel Kir2.

Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase.

C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase. Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain. In contrast to nearly all homodimeric proteins, CHIP is asymmetric.

Dihydropyrimidinones and -thiones with improved activity against human polyomavirus family members.

Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions.

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown.

Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae.

Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.

Loss of Ypk1, the yeast homolog to the human serum- and glucocorticoid-induced protein kinase, accelerates phospholipase B1-mediated phosphatidylcholine deacylation.

Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast.

ESCRT regulates surface expression of the Kir2.1 potassium channel.

Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking.

How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: the early history of ERAD.

All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation.

Hsp70 and Hsp90 multichaperone complexes sequentially regulate thiazide-sensitive cotransporter endoplasmic reticulum-associated degradation and biogenesis.

The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC.

The thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation.

The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation.

Plasmodium falciparum encodes a single cytosolic type I Hsp40 that functionally interacts with Hsp70 and is upregulated by heat shock.

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70-Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P.

Prion-impairing mutations in Hsp70 chaperone Ssa1: effects on ATPase and chaperone activities.

We previously described many Hsp70 Ssa1p mutants that impair [PSI(+)] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1(L483W) ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones.

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer.

In vitro characterization of the Mig1 repressor from Saccharomyces cerevisiae reveals evidence for monomeric and higher molecular weight forms.

The Mig1 DNA-binding protein of Saccharomyces cerevisiae was expressed and purified from yeast and the physical properties were characterized by several methods, including gel filtration, sucrose gradient sedimentation and native gel electrophoresis. Purified Mig1 exists as a monomer with a Stokes' radius of 48 A and a sedimentation coefficient of 3.55 S.

Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner.

Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication.