Biology subjects

Sekhon, H.

Publications and source records attributed to Sekhon, H..

6 recordsLinked to original sources

bioRxiv2026

Activation of PPARγ redirects fibro-adipogenic progenitors to replace ectopic bone with fat in models of fibrodysplasia ossificans progressiva and trauma-induced heterotopic ossification

The pathologic, osteogenic differentiation of fibroadipogenic progenitor cells (FAPs) is the primary recognized contributor to ectopic bone formation in fibrodysplasia ossificans progressiva (FOP) and trauma-induced heterotopic ossification (HO). Both conditions are characterized by up-regulated BMP signaling - the former by a gene mutation rendering the BMP receptor ACVR1 susceptible to activation by inflammatory ligands (Activin A), and the latter by up-regulated presence of BMP2 ligand in the setting of unmutated BMP receptor. We performed an unbiased assessment of FDA-approved therapies which would optimally target the transcriptional aberrations observed in developing FOP and HO lesions based on publicly-available datasets. This analysis uncovered rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonist as the highest scoring therapeutic option across three data sets for both conditions. Rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of FOP, and replaced these lesions with ectopic adipose tissue; similarly, systemic and local rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of trauma-induced HO and replaced these lesions with ectopic adipose tissue. Our findings were corroborated by a single case report from 2010 showing positive results with rosiglitazone in a non-diabetic patient with FOP, with no subsequent studies. Overall, our findings suggest that a previously FDA-approved therapeutic is likely to be a successful therapeutic agent for both FOP and trauma-induced HO, both conditions for which current therapeutic options remain inadequate. One Sentence SummaryWe show that a previously FDA-approved therapeutic known to induce adipogenesis reduces ectopic bone and induces ectopic fat formation in diseases of heterotopic ossification.

cell biology↗

bioRxiv2025

Next Generation of StayGold-based Adaptable Turn-On Maturation (ATOM) Sensors Targeting PSD95, Gephyrin, and HOMER1 Proteins

Genetically encoded biosensors that have the capacity to detect intracellular molecules have proven to be invaluable tools in biology. However, development of biosensors with generalizable binding domains for detecting arbitrary targets of choice is still in its early stages. We previously introduced ATOM biosensor technology in which adaptable monobody and nanobody binding domains are employed for molecular recognition, and biosensor turn-on is achieved by coupling target binding to activation of the attached fluorescent protein by means of conformational change. In this work, we extend the ATOM mechanism to the newly identified, highly photostable fluorescent proteins mStayGold and mBaoJin. We validated the ATOM sensors using three neuronal targets: PSD95, Gephyrin, and HOMER1. The sensors exhibited turn-on ratios of over 100-fold with high specificity. Compared to existing methods for detecting these proteins, ATOM sensors demonstrated improved dynamic range, lower background, and a higher turn-on. The results highlight the adaptable nature of the ATOM mechanism and demonstrate that it allows for development of biosensors with various colors and photophysical characteristics.

synthetic biology↗

bioRxiv2023

Mimicking Kidney Flow Shear Efficiently Induces Aggregation of LECT2, a Protein Involved in Renal Amyloidosis

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress--shear generated by fluid flow through a network of artery and capillary-sized channels--drives LECT2 fibrillogenesis. To mimic blood flow through the human kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 m in width. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering the mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

biochemistry↗

bioRxiv2023

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells

A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro. The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

synthetic biology↗

bioRxiv2022

Engineering Protein Activity into Off-the-Shelf DNA Devices

DNA-based devices are relatively straightforward to design by virtue of their predictable folding, but they lack biological activity. Conversely, protein-based devices offer a myriad of biological functions but are much more difficult to design due to their complex folding. This study bridges the fields of DNA engineering and protein engineering to generate a protein switch that is activated by a specific DNA sequence. A single protein switch, engineered from nanoluciferase using the alternate frame folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for a DNA or RNA sequence of choice, sensors for serotonin and ATP, and a computational device that processes two DNA inputs. nLuc-AFF is a genetically-encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by cell phone camera. nLuc-AFF is not falsely activated by decoy DNA and it retains full ratiometric readout in 100 % serum. The design approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.

synthetic biology↗

bioRxiv2021

Engineering A Fluorescent Protein Color Switch Using Entropy-driven Beta Strand Exchange

Protein conformational switches are widely used in biosensing. They are typically composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational change. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a 6-fold green-to-yellow ratiometric fluorescence change in vitro, and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered GFP variant, to replace one of its {beta}-strands (containing T203 and specifying green fluorescence) with a duplicate {beta}-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This proof-of-concept design has the advantages of full genetic encodability, ratiometric or intensiometric response, and potential for modularity. The latter attribute is enabled by circular permutation of the input domain.

biophysics↗