Biology subjects

Patel, K. J.

Publications and source records attributed to Patel, K. J..

5 recordsLinked to original sources

bioRxiv2026

Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

Cellular stiffness profoundly impacts cancer metastasis at multiple levels, but mechanisms that regulate cancer cells stiffness remain poorly understood. Here, we identified potassium efflux and KCNMB1, an auxiliary subunit of the large conductance potassium efflux (BK) channels, as regulators of cellular stiffness downstream of myocardin related transcription factor A (MRTFA). In primary pericytes, KCNMB1 knockdown increased cellular stiffness, which is consistent with the role of potassium efflux in promoting relaxation during excitation-contraction coupling. In a striking contrast, however, KCNMB1 knockdown decreased cellular stiffness in cancer cells. Softer cancer cells were resistant to NK cell mediated cytotoxicity and the low KCNMB1 expression was associated with worse survival in breast cancer patients. Importantly, pharmacological activation of BK channels reduced metastatic burden in mice and improved lysis of cancer cells by cytotoxic T-lymphocytes. These results highlight the unique ionic regulation of stiffness in cancer cells and point to BK channel agonism as a new therapeutic approach in cancer.

cancer biology↗

bioRxiv2024

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis

Members of the 3-5 RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5-3 RNA and DNA polymerases. Discovery of enzymes capable of this reaction raised the possibility of exploiting 3-5 polymerases for post-transcriptional incorporation of nucleotides to the 5-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other non-coding RNA. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3-5 polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to 8 bases to 5-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5-end labeling tool.

biochemistry↗

bioRxiv2024

Placental cytotrophoblast microvillar stabilization is required for cell-cell fusion

The placenta is an essential organ of pregnancy required for maternal-fetal transport and communication. The surface of the placenta facing the maternal blood is formed by a single giant multinucleate cell: the syncytiotrophoblast. The syncytiotrophoblast is formed and maintained via fusion of progenitor cytotrophoblasts. Cell-cell fusion is a tightly regulated process, and in non-trophoblastic cells is accompanied by stereotypical alterations in cell shape by cells that have attained fusion-competence. The most prominent feature is the formation of actin-based membrane protrusions, but whether stereotypic morphological changes occur in fusion-competent cytotrophoblasts has not been characterized. Using a human placental explant model, we characterized cell shape factors associated with the attainment of cytotrophoblast fusion competence. We found that fusion-competent cytotrophoblasts are hypertrophic, elongated cells, that form microvilli at the apical membrane. The actin-membrane cross linker protein ezrin was found to have highly polarized expression within cytotrophoblast microvilli. Inhibition of ezrin activation destabilized cytotrophoblast microvilli and prevented cytotrophoblast fusion. Thus, we propose that the polarized activation of ezrin within apical microvilli and actin-mediated changes in membrane dynamics are necessary for cytotrophoblast fusion. Summary statementFusion-competent cytotrophoblasts undergo dynamic changes in cell morphology including the acquisition of apically localized microvilli. Microvillar stabilization facilitates effective fusion and differentiation.

developmental biology↗

bioRxiv2023

Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction.

Type I interferons (IFNs) increase the excitability of dorsal root ganglion (DRG) neurons via activation of MNK-eIF4E translation signaling to promote pain sensitization in mice. Activation of STING signaling is a key component of type I IFN induction. Manipulation of STING signaling is an active area of investigation in cancer and other therapeutic areas. Vinorelbine is a chemotherapeutic that activates STING and has been shown to cause pain and neuropathy in oncology clinical trials in patients. There are conflicting reports on whether STING signaling promotes or inhibits pain in mice. We hypothesized that vinorelbine would cause a neuropathic pain-like state in mice via STING and signaling pathways in DRG neurons associated with type I IFN induction. Vinorelbine (10 mg/kg, i.v.) induced tactile allodynia and grimacing in WT male and female mice and increased p-IRF3 and type I IFN protein in peripheral nerves. In support of our hypothesis, vinorelbine-mediated pain was absent in male and female StingGt/Gt mice. Vinorelbine also failed to induce IRF3 and type I IFN signaling in these mice. Since type I IFNs engage translational control via MNK1-eIF4E in DRG nociceptors, we assessed vinorelbine-mediated p-eIF4E changes. Vinorelbine increased p-eIF4E in DRG in WT animals but not in StingGt/Gtor Mknk1-/- (MNK1 KO) mice. Consistent with these biochemical findings, vinorelbine had an attenuated pro-nociceptive effect in male and female MNK1 KO mice. Our findings support the conclusion that activation of STING signaling in the peripheral nervous system causes a neuropathic pain-like state that is mediated by type I IFN signaling to DRG nociceptors.

neuroscience↗

bioRxiv2019

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination during DNA repair

Vertebrate DNA crosslink repair is a crucial process that excises toxic replication-blocking DNA interstrand crosslinks1. This pathway fails in the inherited human disease Fanconi anemia (FA), resulting in abnormal development, loss of blood production, and marked cancer susceptibility2. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause FA3. A biochemical description of how FA gene products might initiate the removal of DNA crosslinks is now emerging. However, structural insight into this vital process has been limited. Here, we use electron cryomicroscopy (cryoEM) to determine the structure of a key DNA crosslink repair factor - FANCD2 heterodimerized with FANCI (D2-I). Recombinant chicken D2-I adopts a conformation that is in agreement with the structure of its murine counter-part4. In contrast, the activated monoubiquitinated form of D2-I (ubD2-I) adopts an alternative closed conformation, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a molecular pin to trap ubD2-I in the closed conformation, clamped on DNA. The new solvent-exposed interface around the monoubiquitination site is likely a platform for recruitment of other DNA repair factors. We also find that isolated FANCD2 is a dimer, but it adopts a closed conformation that is unable to bind DNA. When incubated with free FANCI, the FANCD2 homodimer exchanges to D2-I, acquiring DNA binding properties. This suggests an autoinhibitory mechanism that prevents FANCD2 activation on DNA until after assembly with FANCI. Together, our cryoEM and biochemical analysis suggests that D2-I is a clamp that is locked onto DNA by ubiquitin, and provides unanticipated new insight into the regulation and the initiation of DNA cross-link repair.

molecular biology↗