Biology subjects

Sahasrabuddhe, A.

Publications and source records attributed to Sahasrabuddhe, A..

3 recordsLinked to original sources

bioRxiv2025

Novel mechanistic insights for catalytic bioluminescence of mammalian Gaussia Luciferase through mutant and ancestral analysis

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized E. Coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored fluorescent coelenerazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The non-luminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover, and that this impacts the flash-type luminescence of the luciferase; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography on luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

biochemistry↗

bioRxiv2020

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via -helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by -helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

synthetic biology↗

bioRxiv2020

Tyrosine phosphorylation of ACLY regulates lipid metabolism and oncogenesis in ALK positive Anaplastic Large Cell Lymphoma

A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased {beta}-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (p<0.01) and +18-oleyol carnitine (p<0.001). Similarly, oxygen consumption rate (OCR) is significantly increased in cells expressing ACLY-Y682F (p<0.001). Moreover, expression of ACLY-Y682F dramatically decreases cell proliferation, impairs clonogenicity and abrogates tumor growth in vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

cancer biology↗