Biology subjects

Shah, R. R.

Publications and source records attributed to Shah, R. R..

3 recordsLinked to original sources

bioRxiv2023

Structure-guided design and optimization of covalent VHL-targeted sulfonyl fluoride PROTACs

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/545773v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@da22e5org.highwire.dtl.DTLVardef@1290278org.highwire.dtl.DTLVardef@1611c0eorg.highwire.dtl.DTLVardef@7902d2_HPS_FORMAT_FIGEXP M_FIG C_FIG Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest, however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1 binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or androgen receptor (AR) without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design expanding the substrate scope of covalent E3 ligase PROTACs.

molecular biology↗

bioRxiv2023

Endogenous Tenocyte Activation Underlies the Regenerative Capacity of Adult Zebrafish Tendon

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that the zebrafish tendon can regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). We show that tendon regeneration progresses in three main phases: inflammation within 24 hours post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration. Collectively, our work debuts the zebrafish tendon as one of the only reported adult tendon regenerative models and positions it as an invaluable comparative system to identify regenerative mechanisms that may inspire new tendon injury treatments in the clinic.

developmental biology↗

bioRxiv2018

A robust method for RNA extraction and purification from a single adult mouse tendon

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.\n\nMethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.\n\nResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.\n\nDiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

molecular biology↗