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Tripathi, Sarvind

Publications and source records attributed to Tripathi, Sarvind.

10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A tyrosine phosphoregulatory system controls exopolysaccharide biosynthesis and biofilm formation in Vibrio cholerae

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch

Post-translational control of PERIOD stability by Casein Kinase 1δ and ε (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the kinase activation loop and determines which sites on mammalian PER2 are preferentially phosphorylated, thereby directly regulating PER2 stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.

59 BASIC BIOLOGICAL SCIENCES↗