Biology subjects

Parvin, J. D.

Publications and source records attributed to Parvin, J. D..

5 recordsLinked to original sources

bioRxiv2024

Identification of TAP2 protein variants resistant to inhibition by the HSV1 ICP47 protein

Herpes Simplex Virus 1 evades the host immune system by expressing a protein, ICP47, that binds to and inhibits the heterodimeric Transporter Associated with Antigen Processing (TAP). We screened a library of 1786 variants in TAP2, one of the components of the TAP heterodimer, and identified 39 variants that were resistant to inhibition by ICP47. Of these 39 variants, five were individually tested, and three (T257I, S274H, and T244R) were confirmed to be significantly resistant to inhibition by ICP47. This resistance to inhibition did not extend to the Epstein Barr Virus BNLF2a protein, another viral factor known to inhibit antigen presentation by targeting TAP. These three residues localize close to the binding site of ICP47, on the 3D structure of TAP, but only Ser274 is spatially close to the antigenic peptide binding site of TAP. These results functionally resolve the TAP2 residues required for peptide binding from those required for ICP47 binding and identify TAP2 residues whose targeting with small molecule inhibitors could effectively prevent Herpes virus downregulation of antigen processing. AUTHOR SUMMARYT cells detect virus infected cells via viral peptides in complex with the major histocompatibility complex type I (MHC-I) on the cell surface. Herpes Simplex Virus 1 expression of the ICP47 protein allows the virus to evade detection and clearance of infected cells by host T cells. ICP47 binds to the TAP1/TAP2 protein complex and prevents the loading of antigenic peptides onto the MHC-I protein complex and thereby blocks the antigen processing. In this study, we screened for and identified variants of TAP2 with single amino acid substitutions that are no longer inhibited by ICP47. TAP2 proteins with the identified amino acid substitutions functioned normally in antigen processing even in the presence of ICP47. Results from this study create the potential strategy for drugs that target the identified portions of the TAP2 protein and block ICP47 inhibition of antigen processing while retaining normal TAP1/TAP2 function and thus suggest a new antiviral therapeutic approach for herpes virus infections.

immunology↗

bioRxiv2024

Multiplexed functional analysis of TAP2 variants in regulating MHC-I cell surface abundance reveals overexpression of PLK1 downregulates antigen presentation.

The abundance of MHC-I on the cell surface depends on its association with antigenic peptides transported to the endoplasmic reticulum by the Transporter Associated with Antigen Processing (TAP), a peptide channel composed of TAP1 and TAP2. We functionally screened over 1400 TAP2 variants for effects on MHC-I cell surface abundance. Amino-acid substitutions of loss of function (LOF) variants clustered in the NTP-binding domain and along the TAP1-TAP2 binding interface, suggesting that these variants interfered with TAP conformational changes associated with peptide transport. Some LOF variants carried potential phosphomimetic substitutions; one such substitution at Ser251 was embedded in a sequence context consistent with the phosphorylation motif of PLK kinases. Inhibition of PLK kinases increased MHC-I surface expression, while overexpression of PLK1 in cells decreased MHC-I in wild type TAP2, but not in TAP2-S251A-expressing cells. Importantly, site-specific phosphorylation of TAP2 in human tumor samples correlated with alterations of gene expression in the cell cycle and antigen presentation pathways, consistent with the notion that phosphorylation downregulates antigenic peptide transport in human tumors. These data strongly support the hypothesis that in cancer cells, TAP2 phosphorylation by PLK1, and perhaps other kinases, can downregulate the antigen presentation process.

cancer biology↗

bioRxiv2023

DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1. AUTHOR SUMMARYMost missense substitutions in BRCA1 are variants of unknown significance (VUS), and individuals with a VUS in BRCA1 cannot know from genetic information alone whether this variant predisposes to breast or ovarian cancer. We apply a multiplexed functional assay for homology directed repair of DNA double strand breaks to assess variant impact on this important BRCA1 protein function. We analyzed 2172 variants in the amino-terminus of BRCA1 and demonstrate that variants that are known as pathogenic have a loss of function in the DNA repair assay. Conversely, variants that are known to be benign are functionally normal in the multiplexed assay. We suggest that these functional determinations of BRCA1 variants can be used to augment the information that clinical cancer geneticists provide to patients who have a VUS in BRCA1.

cancer biology↗

bioRxiv2020

Modulation of Early Mitotic Inhibitor 1 (EMI1) Depletion on the Sensitivity of PARP Inhibitors in BRCA1 Mutated Triple-Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) represents approximately 10-15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 inhibitor. Surprisingly, EMI1 depletion also led to resistance to a MEK inhibitor, though this inhibitor blocks cells in G1 phase of the cell cycle and would not be expected to be sensitive to EMI1 levels. Notably, increasing the RAD51 protein expression only partially recapitulated the effects of EMI1 depletion in causing resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking mitotic entry. We found that combining CHK1 inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.

cancer biology↗

bioRxiv2018

A multiplexed homology-directed DNA repair assay reveals the impact of ~1,700 BRCA1 variants on protein function

Loss-of-function mutations in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for mutations in the BRCA1 gene frequently reveals a missense variant for which the impact on the molecular function of the BRCA1 protein is unknown. Functional BRCA1 is required for homology directed repair (HDR) of double-strand DNA breaks, a key activity for maintaining genome integrity and tumor suppression. Here we describe a multiplex HDR reporter assay to simultaneously measure the effect of hundreds of variants of BRCA1 on its role in DNA repair. Using this assay, we measured the effects of ~1,700 amino acid substitutions in the first 302 residues of BRCA1. Benchmarking these results against variants with known effects, we demonstrate accurate discrimination of loss-of-function versus benign variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

genomics↗