Biology subjects

Camacho, C. V.

Publications and source records attributed to Camacho, C. V..

5 recordsLinked to original sources

bioRxiv2024

Development and Characterization of Recombinant ADP-Ribose Binding Reagents that Allow Simultaneous Detection of Mono and Poly ADP-Ribose

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADP-ribosylation. Recently we developed a set of recombinant antibody-like ADP-ribose binding proteins, using naturally occurring ADPR binding domains (ARBDs) that include macrodomains and WWE domains, that have been functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with two other species, the Fc for mouse and goat immunogloblulin. Characterization of the new reagents indicates that they can be detected in a species-dependent manner, recognize specific ADP-ribose moieties, and excitingly, can be used in various antibody-based assays by co-staining. The expansion of this tool will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

molecular biology↗

bioRxiv2024

Relaxin Modulates the Genomic Actions and Biological Effects of Estrogen in the Myometrium by Reducing Estrogen Receptor Alpha Phosphorylation

Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ER). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explored the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild-type or mutant ER (hTERT-HM-ER cells). Our results indicate that Rln modulates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation of ER on serine 118 (S118), as well as by reducing the E2-dependent binding of ER across the genome. These effects were associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of some genes and enhanced expression of others. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ER required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ER signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes.

molecular biology↗

bioRxiv2023

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3{eta}, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells. SummaryWe have discovered a druggable PARP14/TARG1-regulated pathway that mediates site- specific mono(ADP-ribosyl)ation of RACK1, a ribosomal protein. This pathway controls stress granule assembly and disassembly, as well as the translation of a subset of mRNAs, to modulate the growth of ovarian cancer cells in culture and in vivo.

cell biology↗

bioRxiv2022

Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers

AO_SCPLOWBSTRACTC_SCPLOWLong non-coding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression of lncRNA152 (lnc152; a.k.a. DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown of lnc152 promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression of lnc152 inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice, lnc152 inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated that lnc152 downregulates genes controlling angiogenesis. Using pull down assays coupled with LC-MS/MS, we identified RBM47, a known tumor suppressor in breast cancer, as a lnc152-interacting protein. The effects of lnc152 in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate that lnc152 is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC. SO_SCPLOWTATEMENTC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWOFC_SCPLOW SO_SCPLOWIGNIFICANCEC_SCPLOWThis study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC.

cancer biology↗

bioRxiv2021

Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose)

AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems.

synthetic biology↗