Biology subjects

Shinde, U.

Publications and source records attributed to Shinde, U..

4 recordsLinked to original sources

bioRxiv2024

An effective method of measuring nanobody binding kinetics and competition-based epitope mapping using biolayer interferometry

Protein-protein interactions (PPI) underpin nearly all biological processes, and understanding the molecular mechanisms governing these interactions is crucial for the progress of biomedical sciences. The emergence of AI-driven computational tools can help reshape the methods in structural biology, however model data often quires empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of PPIs. Biolayer interferometry (BLI) is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here, we provide step-by-step instructions on how to perform kinetics experiments using BLI. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 hours to complete and is suitable for users with minimal experience with biochemical techniques.

biochemistry↗

bioRxiv2023

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

biochemistry↗

bioRxiv2022

Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors

Chromosomal rearrangements of ROS1 generate ROS1 tyrosine kinase fusion proteins that are established oncogenes predicting effectiveness of tyrosine kinase inhibitors (TKI) treatment. The cancer genome reveals nonsynonymous missense mutations in ROS1, however, their oncogenic potential remains unknown. We nominated thirty-four tumor-associated missense mutations in ROS1 kinase domain for functional interrogation. Immunoblotting revealed diverse impact of the mutations on the kinase, ranging from loss of function to significant increase in catalytic activity. Notably, Asn and Gly substitutions at the Asp-2113 position in ROS1 kinase domain were TKI- sensitive hyper-activating mutations, and transformative oncogenes in independent cell models. Molecular modeling revealed drastic alterations in the activation loop of ROS1D2113N compared to wildtype kinase. Proteomics studies showed that ROS1D2113N increases phosphorylation of known effectors akin to ROS1 fusions, and upregulates pathways not previously linked to ROS1, including mTORC2, JNK1/2, AP-1, TGFB1 and CCN1/2. In vivo, ROS1D2113N drove tumor formation that was sensitive to inhibition by crizotinib and lorlatinib. Taken together, these data show that select point mutations within ROS1 RTK are oncogenic, and maybe therapeutically targetable with FDA-approved TKI.

cancer biology↗

bioRxiv2022

A set of orthogonal versatile interacting peptide tags for imaging cellular proteins

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a proteins behavior in situ. Some proteins can be analyzed by fusion to protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in living cells. Labeling is mediated by the formation of a high affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific, with no cross-reactivity between the three VIP tags under cellular conditions. Lastly, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin), highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

biochemistry↗