Biology subjects

Gaur, D.

Publications and source records attributed to Gaur, D..

6 recordsLinked to original sources

bioRxiv2025

An Msp1-Protease Chimera Captures Transient AAA+ Interactions and Unveils Ost4 Mislocalization Errors

Membrane protein homeostasis (proteostasis) is essential for maintaining the integrity of eukaryotic organelles. Msp1 is a membrane anchored AAA+ (ATPase Associated with cellular Activities) protein that maintains mitochondrial proteostasis by extracting aberrant proteins from the outer mitochondrial membrane. A comprehensive understanding of the physiological roles of Msp1 has been hindered because AAA+ proteins interact with substrates transiently and common strategies to stabilize this interaction lead to undesirable mitochondrial phenotypes. To circumvent these drawbacks, we fused catalytically active Msp1 to the inactivated protease domain of the AAA+ protease Yme1. The resulting chimera sequesters substrates in the catalytically inactive degradation chamber formed by the protease domain. We performed mass spectrometry analysis with the Msp1-protease chimera and identified the signal anchored protein Ost4 as a novel Msp1 substrate. Topology experiments show that Ost4 adopts mixed orientations when mislocalized to mitochondria and that Msp1 extracts mislocalized Ost4 regardless of orientation. Together, this work develops new tools for capturing transient interactions with AAA+ proteins, identifies new Msp1 substrates, and shows a surprising error in targeting of Ost4.

biochemistry↗

bioRxiv2025

Defects in nephrogenesis result in an expansion of the Foxd1+ stromal progenitor population

Reciprocal signaling interactions coordinate multiple aspects of kidney development. While signals from the stroma have been shown to regulate nephron progenitor cell (NPC) differentiation, much less is known about regulation of the stromal progenitor population. Here, we demonstrate that disruption of the NPC lineage via loss of Wt1 (i.e., Six2cre;Wt1c/c) results in an expansion of Foxd1+ stromal progenitor cells. Analyses of the developing stroma in two additional models, including Wnt4-null mutants (which fail to form nephron structures similar to Six2cre;Wt1c/c kidneys) and NPC ablation via diphtheria toxin (i.e., Six2cre;RosaDTAc/+), both phenocopy Six2cre;Wt1c/c mutants, thus further confirming that defects in the NPC lineage result in abnormal development of the stromal progenitor population. Furthermore, we identify a subcluster of the Foxd1+ stroma that appears expanded in the three mutant mouse models and conserved in human fetal kidneys. Overall, the findings from this study suggest that loss of differentiating nephron structures may result in possible over proliferation of the stromal progenitor population and/or a block in stromal differentiation and further highlight how crosstalk amongst the progenitor cell lineages coordinates multiple aspects of kidney development. KEY POINTSO_LIMutant mouse models targeting the nephron lineage (i.e., Six2cre;Wt1c/c and Wnt4-null mutants which fail to form early nephron structures) suggest that a block in NPC differentiation results in an abnormal expansion of the Foxd1+ stromal progenitor population. C_LIO_LIMutant kidneys with NPC ablation (i.e., Six2cre;RosaDTAc/+) show maintenance of stromal progenitors independent of signals from adjacent nephron progenitors and ureteric bud. C_LIO_LISingle nuclei RNA-seq identifies three subclusters of the Foxd1+ stromal progenitor population at E15.5, including one cluster of proliferating cells and a distinct Fap+, Igf1+, Gria1+, Gdnf- subcluster which appears expanded in Six2cre;Wt1c/c, Wnt4-null, and Six2cre;RosaDTAc/+ mutant kidneys and conserved in human fetal kidneys. C_LI

developmental biology↗

bioRxiv2025

A suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression

Expression and purification of recombinant proteins in E. coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C-terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a completely scarless vector series with 9 different N-terminal tags. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.

biochemistry↗

bioRxiv2024

Energetic requirements and mechanistic plasticity in Msp1-mediated substrate extraction from lipid bilayers

AAA+ proteins are essential molecular motors involved in numerous cellular processes, yet their mechanism of action in extracting membrane proteins from lipid bilayers remains poorly understood. One roadblock for mechanistic studies is the inability to generate subunit specific mutations within these hexameric proteins. Using the mitochondrial AAA+ protein Msp1 as a model, we created covalently linked dimers with varying combinations of wild type and catalytically inactive E193Q mutations. The wide range of ATPase rates in these constructs allows us to probe how Msp1 uses the energy from ATP hydrolysis to perform the thermodynamically unfavorable task of removing a transmembrane helix (TMH) from a lipid bilayer. Our in vitro and in vivo assays reveal a non-linear relationship between ATP hydrolysis and membrane protein extraction, suggesting a minimum ATP hydrolysis rate is required for effective TMH extraction. While structural data often supports a sequential clockwise/2-residue step (SC/2R) mechanism for ATP hydrolysis, our biochemical evidence suggests mechanistic plasticity in how Msp1 coordinates ATP hydrolysis between subunits, potentially allowing for robustness in processing challenging substrates. This study enhances our understanding of how Msp1 coordinates ATP hydrolysis to drive mechanical work and provides foundational insights about the minimum energetic requirements for TMH extraction and the coordination of ATP hydrolysis in AAA+ proteins.

biochemistry↗

bioRxiv2023

The AAA+ protein Msp1 selects substrates by recognizing a hydrophobic mismatch between the substrate transmembrane domain and the lipid bilayer

An essential aspect of protein quality control is enzymatic removal of membrane proteins from the lipid bilayer. Failures in this essential cellular process are associated with neurodegenerative diseases and cancer. Msp1 is a AAA+ (ATPases Associated with diverse cellular Activities) protein that removes mistargeted proteins from the outer mitochondrial membrane (OMM). How Msp1 selectively recognizes and extracts substrates within the complex OMM ecosystem, and the role of the lipid bilayer on these processes is unknown. Here, we describe the development of fully defined, rapid, and quantitative extraction assay that retains physiological substrate selectivity. Using this new assay, we systematically modified both substrates and the lipid environment to demonstrate that Msp1 recognizes substrates by a hydrophobic mismatch between the substrate TMD and the lipid bilayer. We further demonstrate that the rate limiting step in Msp1 activity is extraction of the TMD from the lipid bilayer. Together, these results provide foundational insights into how the lipid bilayer influences AAA+ mediated membrane protein extraction.

biochemistry↗

bioRxiv2021

Plasmodium falciparum and helminth coinfections increase IgE and parasite-specific IgG responses

Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites since they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (p[≤] 0.05). There was a positive association between exposure/infection to P. falciparum and exposure/infection to helminths or the number of helminth species, and vice versa (p[≤] 0.001). In addition, children coexposed/coinfected tended (p= 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/445753v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@a1dc3corg.highwire.dtl.DTLVardef@82128corg.highwire.dtl.DTLVardef@18fcd98org.highwire.dtl.DTLVardef@2d5bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗