Biology subjects

Brodsky, J. L.

Publications and source records attributed to Brodsky, J. L..

7 recordsLinked to original sources

bioRxiv2024

A new series of fluorogen-activating proteins for quantitative protein trafficking and co-localization studies in S. cerevisiae

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

cell biology↗

bioRxiv2024

Excess dietary sodium partially restores salt and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron

The maintenance of fluid and electrolyte homeostasis by the kidney requires proper folding and trafficking of ion channels and transporters in kidney epithelia. Each of these processes requires a specific subset of a diverse class of proteins termed molecular chaperones. One such chaperone is GRP170, which is an Hsp70-like, endoplasmic reticulum (ER)-localized chaperone that plays roles in protein quality control and protein folding in the ER. We previously determined that loss of GRP170 in the mouse nephron leads to hypovolemia, electrolyte imbalance, and rapid weight loss. In addition, GRP170-deficient mice develop an AKI-like phenotype, typified by tubular injury, elevation of clinical kidney injury markers, and induction of the unfolded protein response (UPR). By using an inducible GRP170 knockout cellular model, we confirmed that GRP170 depletion induces the UPR, triggers an apoptotic response, and disrupts protein homeostasis. Based on these data, we hypothesized that UPR induction underlies hyponatremia and volume depletion in rodents, but that these and other phenotypes might be rectified by supplementation with high salt. To test this hypothesis, control and GRP170 tubule-specific knockout mice were provided with a diet containing 8% sodium chloride. We discovered that sodium supplementation improved electrolyte imbalance and reduced clinical kidney injury markers, but was unable to restore weight or tubule integrity. These results are consistent with UPR induction contributing to the kidney injury phenotype in the nephron-specific GR170 knockout model, and that the role of GRP170 in kidney epithelia is essential to both maintain electrolyte balance and cellular protein homeostasis.

cell biology↗

bioRxiv2024

Defining a small-molecule stimulator of the human Hsp70-disaggregase system with selectivity for DnaJB proteins

Hsp70, Hsp40, and Hsp110 form a human protein-disaggregase system that solubilizes and reactivates proteins trapped in aggregated states. However, this system fails to maintain proteostasis in fatal neurodegenerative diseases. Here, we potentiate the human Hsp70-disaggregase system pharmacologically. By scouring a collection of dihydropyrimidines, we disambiguate a small molecule that specifically stimulates the Hsp70-disaggregase system against disordered aggregates and -synuclein fibrils. The newly identified lead compound stimulates the disaggregase activity of multiple active human Hsp70, Hsp40, Hsp110 chaperone sets, with selectivity for combinations that include DnaJB1 or DnaJB4 as the Hsp40. We find that the relative stoichiometry of Hsp70, Hsp40, and Hsp110 dictates disaggregase activity. Remarkably, our lead compound shifts the composition of active chaperone stoichiometries by preferentially activating combinations with lower DnaJB1 concentrations. Our findings unveil a small molecule that stimulates the Hsp70-disaggregase system, even at suboptimal chaperone stoichiometries, which could be developed for the treatment of neurodegenerative diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/575109v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f1b574org.highwire.dtl.DTLVardef@1bfd3aborg.highwire.dtl.DTLVardef@e191e3org.highwire.dtl.DTLVardef@130d19c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

FITM2 deficiency results in ER lipid accumulation, ER stress, reduced apolipoprotein B lipidation, and VLDL triglyceride secretion in vitro and in mouse liver

Structured AbstractO_ST_ABSObjectivesC_ST_ABSTriglyceride (TG) association with apolipoprotein B100 (apoB100) serves to form very low density lipoproteins (VLDL) in the liver. The repertoire of factors that facilitate this association is incompletely defined. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytoplasmic lipid droplets (LDs) in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. MethodsExperiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, simulated Raman spectroscopy (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to LDL density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. Principal conclusionsThe results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be a limiting factor that ultimately contributes to non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH).

biochemistry↗

bioRxiv2023

Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum functions

GRP170, a product of the Hyou1 gene, is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds non-native proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of embryonic fibroblasts from mice in which LoxP sites were engineered in the Hyou1 loci (Hyou1LoxP/LoxP). A doxycycline-regulated Cre recombinase was also stably introduced into these cells. Induction of Cre resulted in excision of Hyou1 and depletion of Grp170 protein, culminating in apoptotic cell death. As Grp170 levels fell we observed increased steady-state binding of BiP to a client, slowed degradation of a misfolded BiP substrate, and BiP accumulation in NP40-insoluble fractions. Consistent with disrupted BiP functions, we observed reactivation of BiP storage pools and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and insights into mutations in the Hyou1 locus and human disease.

cell biology↗

bioRxiv2023

Genome mining yields new disease-associated ROMK variants with distinct defects

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal. Although there is no cure for this disease, specific genes that lead to different Bartter syndrome subtypes have been identified. Bartter syndrome type II specifically arises from mutations in the KCNJ1 gene, which encodes the renal outer medullary potassium channel, ROMK. To date, over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified. Yet, their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined ROMK genomic data in both the NIH TOPMed and ClinVar databases with the aid of a computational algorithm that predicts protein misfolding and disease severity. Subsequent phenotypic studies using a high throughput yeast screen to assess ROMK function--and analyses of ROMK biogenesis in yeast and human cells--identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced protein expression at the cell surface. Another ERAD-targeted ROMK mutant (L320P) was found in only one of the screens. In contrast, another mutation (T300R) was ERAD-resistant, but defects in ROMK activity were apparent after expression and two-electrode voltage clamp measurements in Xenopus oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies. Author SummaryBartter syndrome is a rare genetic disorder characterized by defective renal electrolyte handing, leading to debilitating symptoms and, in some patients, death in infancy. Currently, there is no cure for this disease. Bartter syndrome is divided into five types based on the causative gene. Bartter syndrome type II results from genetic variants in the gene encoding the ROMK protein, which is expressed in the kidney and assists in regulating sodium, potassium, and water homeostasis. Prior work established that some disease-associated ROMK mutants misfold and are destroyed soon after their synthesis in the endoplasmic reticulum (ER). Because a growing number of drugs have been identified that correct defective protein folding, we wished to identify an expanded cohort of similarly misshapen and unstable disease-associated ROMK variants. To this end, we developed a pipeline that employs computational analyses of human genome databases with genetic and biochemical assays. Next, we both confirmed the identity of known variants and uncovered previously uncharacterized ROMK variants associated with Bartter syndrome type II. Further analyses indicated that select mutants are targeted for ER-associated degradation, while another mutant compromises ROMK function. This work sets-the-stage for continued mining for ROMK loss of function alleles as well as other potassium channels, and positions select Bartter syndrome mutations for correction using emerging pharmaceuticals.

genetics↗

bioRxiv2018

Proteome-scale detection of drug-target interactions using correlations in transcriptomic perturbations

The development of an expanded chemical space for screening is an essential step in the challenge of identifying chemical probes for new, genomic-era protein targets. However, the difficulty of identifying targets for novel compounds leads to the prioritization of synthesis linked to known active scaffolds that bind familiar protein families, slowing the exploration of available chemical space. To change this paradigm, we validated a new pipeline capable of identifying compound-protein interactions even for compounds with no similarity to known drugs. Based on differential mRNA profiles from drug treatments and gene knockdowns across multiple cell types, we show that drugs cause gene regulatory network effects that correlate with those produced by silencing their target protein-coding gene. Applying supervised machine learning to exploit compound-knockdown signature correlations and enriching our predictions using an orthogonal structure-based screen, we achieved top-10/top-100 target prediction accuracies of 26%/41%, respectively, on a validation set 152 FDA-approved drugs and 3104 potential targets. We further predicted targets for 1680 compounds and validated a total of seven novel interactions with four difficult targets, including non-covalent modulators of HRAS and KRAS. We found that drug-target interactions manifest as gene expression correlations between drug treatment and both target gene knockdown and up/down-stream knockdowns. These correlations provide biologically relevant insight on the cell-level impact of disrupting protein interactions, highlighting the complex genetic phenotypes of drug treatments. Our pipeline can accelerate the identification and development of novel chemistries with potential to become drugs by screening for compound-target interactions in the full human interactome.

genomics↗