Biology subjects

Uppalapati, M.

Publications and source records attributed to Uppalapati, M..

5 recordsLinked to original sources

bioRxiv2025

An ultra-low background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain

The robustness and broad applicability of an optogenetic tool depends heavily on the properties of the underlying photoreceptor protein and its cognate binding partner - the light responsive core. Current red light optogenetic systems for use in mammalian cells all rely on phytochrome based photoreceptors. These are large (70 kDa) proteins that act as dimers, thereby enforce dimerization on attached proteins. Naturally occurring or engineered binding partners can function effectively in certain cases, but large size, complex mode of interaction, background binding, relatively weak affinity and/or low fold changes between on and off states are significant limitations. Using structure-based design and directed evolution we developed a small (17 kDa) monomeric bilverdin binding photoreceptor FenixS, and a highly selective, high-affinity binder, Ash1 (6 kDa). Negligible off-state binding and a >1200-fold increase in binding affinity upon 700 nm illumination result in a high performance, ultra-low background, light responsive core for a diverse range of applications. An optogenetic tool for red light activation of transcription in mammalian cells based on the FenixS-Ash1 core exhibits robust performance without the need for biliverdin supplementation.

synthetic biology↗

bioRxiv2025

Red-light-only control of protein-protein interactions using a cyanobacteriochrome (UNICYCL)

Most optogenetic tools are controlled by blue light. Red-light responsive tools enable multiwavelength applications and allow greater biological tissue penetration with reduced toxicity. Current red-light tools are primarily based on phytochromes, large dimeric proteins with a structurally complex mode of interaction with their binding partners. Here we introduce a small red-light-only responsive system composed of a GA domain (6 kDa) that binds to a CBCR GAF domain (17 kDa) with a Kd [~]1-5 {micro}M to form a 1:1 complex in the dark. Red light causes dissociation of the complex by causing a >25-fold decrease in binding affinity. The CBCR GAF domain reverts to the dark state with a half-life of [~]1 min and the complex reforms. Structural analysis using NMR measurements combined with molecular docking and dynamics simulations shows that the binder interacts with the GAF domain and senses isomerization of the bilin chromophore at a site that overlaps the critical tongue domain of phytochromes. This system provides a small, simple red-light-only optogenetic tool that can operate to control protein-protein interactions in vitro and in living cells.

biochemistry↗

bioRxiv2024

Zr-labeled N4MU01 radioimmunoconjugates as theranostics against nectin-4 positive triple negative breast cancer

PurposeNectin-4 is an overexpressed biomarker in 60-70% of triple-negative breast cancer (TNBC), and an ideal target for radiotherapy and PET imaging. In this study, we have developed theranostic radioimmunoconjugates (RICs) based on a fully-human anti-nectin-4 antibody, N4MU01. We characterized and evaluated the efficacy of these RICs for applications in molecular imaging and radiotherapy of aggressive TNBC models. MethodsAn anti-nectin-4 antibody (N4MU01) was radiolabeled with 89Zr and 225Ac, for imaging and radiotherapy, respectively, using TNBC xenograft and syngeneic models. Biodistribution & PET imaging of [89Zr]Zr-DFO-N4MU01 RIC was studied in mice bearing nectin-4 positive xenografts. Dosimetry of [225Ac]Ac-Macropa-N4MU01 was studied in healthy mice, and therapeutic efficacy was evaluated in mice bearing human TNBC MDA-MB-468 xenograft and in a syngeneic xenograft model using murine 4T1 breast cancer cells transfected with human nectin-4 (4T1.nectin-4). Mice received 2 doses of 13 kBq or 18.6 kBq 10 days apart. ResultsThe pharmacokinetic profile of [89Zr]Zr-DFO-N4MU01 RIC showed biphasic distribution with a moderate elimination half-life of 63 h. PET imaging and biodistribution of [89Zr]Zr-DFO-N4MU01 in mice bearing MDA-MB-468 xenograft showed high tumor uptake of 13.2 {+/-} 1.12 %IA/g at 120 h. [225Ac]Ac-Macropa-N4MU01 was effectively internalized in MDA-MB-468 and was cytotoxic to the cells with IC50 of 1.2 kBq/mL. Mice bearing MDA-MB-468 xenograft treated with [225Ac]Ac-Macropa-N4MU01 (13 kBq or 18.6 kBq) had a remarkable tumor growth inhibition that was dose dependent. For the syngeneic 4T1.nectin-4 model, treatment with [225Ac]Ac-Macropa-N4MU01 (13 kBq) led to complete tumor remission in 83.3% (5/6) of mice. ConclusionThe specific tumor uptake and remarkable effectiveness at shrinking aggressive TNBC tumors is very promising towards clinical development of N4MU01 RICs as theranostics against TNBC.

pharmacology and toxicology↗

bioRxiv2022

AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically-polarized, evolutionarily-conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but little is known about the molecular regulation of planar divisions. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN, and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light into how spindle orientation influences epidermal stratification.

developmental biology↗

bioRxiv2019

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

Existing optogenetic tools for controlling protein-protein interactions are available in a limited number of wavelengths thereby limiting opportunities for multiplexing. The cyanobacteriochrome (CBCR) family of photoreceptors responds to an extraordinary range of colors, but light-dependent binding partners for CBCR domains are not currently known. We used a phage-display based approach to develop small (~50-residue) monomeric binders selective for the green absorbing state (Pg), or for the red absorbing state (Pr) of the CBCR Am1_c0023g2 with a phycocyanobilin chromophore and also for the far-red absorbing state (Pfr) of Am1_c0023g2 with a biliverdin chromophore. These bind in a 1:1 mole ratio with KDs for the target state from 0.2 to 2 M and selectivities from 10 to 500-fold. We demonstrate green, orange, red, and far-red light-dependent control of protein-protein interactions in vitro and also in vivo where these multicolor optogenetic tools are used to control transcription in yeast.

synthetic biology↗