Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

A Potent, Selective CBX2 Chromodomain Ligand and Its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation.

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Exploring the interaction between Mycobacterium tuberculosis enolase and human plasminogen using computational methods and experimental techniques.

Surface localized microbial enolases' binding with human plasminogen has been increasingly proven to have an important role in initial infection cycle of several human pathogens. Likewise, surface localized Mycobacterium tuberculosis (Mtb) enolase also binds to human plasminogen, and this interaction may entail crucial consequences for granuloma stability. The current study is the first attempt to explore the plasminogen interacting residues of enolase from Mtb. Beginning with the structural modeling of Mtb enolase, the binding pose of Mtb enolase and human plasminogen was predicted using protein-protein docking simulations. The binding pose revealed the interface region with interacting residues and molecular interactions. Next, the interacting residues were refined and ranked by using various criteria. Finally, the selected interacting residues were tested experimentally for their involvement in plasminogen binding. The two consecutive lysine residues, Lys-193 and Lys-194, turned out to be active residues for plasminogen binding. These residues when substituted for alanine along with the most active residue Lys-429, that is, the triple mutant (K193A + K194A + K429A) Mtb enolase, exhibited 40% reduction in plasminogen binding. It is worth noting that Mtb enolase lost nearly half of the plasminogen binding activity with only three simultaneous substitutions, without any significant secondary structure perturbation. Further, the sequence comparison between Mtb and human enolase isoforms suggests the possibility of selective targeting of Mtb enolase to obstruct binding of human plasminogen.

Enolase of Mycobacterium tuberculosis is a surface exposed plasminogen binding protein.

BACKGROUND: Enolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. METHODS: Anti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. RESULTS: Enolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TB patients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with Mtb H37Rv. CONCLUSIONS: Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. GENERAL SIGNIFICANCE: Plasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein.

Overexpression of the pleiotropic regulator CodY decreases sporulation, attachment and pellicle formation in Bacillus anthracis.

CodY, a global transcriptional regulator, primarily functions as a nutrient and energy sensor. It is activated by metabolic effectors like BCAA and GTP. In low G + C Gram positive bacteria, it facilitates coupling of changes in the cellular metabolite pool with those required in the transcriptome of the cell. This pleiotropic regulator controls the expression of a vast number of genes as the cell transits from exponential to the stationary phase. Earlier studies have shown that CodY is required for the virulence of Bacillus anthracis. We sought to investigate the effect of its overexpression on the physiology of B. anthracis. In our study, we found that cellular CodY levels were unchanged during this phase-transition. Expression of endogenous CodY remained the same in different nutrient limiting conditions. Immunoblotting studies revealed CodY presence in the whole spore lysate of B. anthracis indicating it to be a component of the spore proteome. We could also detect CodY in the secretome of B. anthracis. Further, CodY was overexpressed in B. anthracis Sterne strain and this led to a 100-fold decrease in the sporulation titer and a 2.5-fold decrease in the in vitro attachment ability of the bacteria. We also observed a decrease in the pellicle formation by CodY overexpressed strain when compared to wildtype bacilli. The CodY overexpressed strain showed chaining phenotype during growth in liquid media and pellicle.

WalRK two component system of Bacillus anthracis responds to temperature and antibiotic stress.

WalRK Two Component System (TCS) of Bacillus anthracis forms a functional TCS. This report elaborates upon the WalRK genomic architecture, promoter structure, promoter activity and expression under various stress conditions in B. anthracis. 5' RACE located the WalRK functional promoter within 317 bp region upstream of WalR. Reporter gene assays demonstrated maximal promoter activity during early growth phases indicating utility in exponential stages of growth. qRT-PCR showed upregulation of WalRK transcripts during temperature and antibiotic stress. However, WalR overexpression did not affect the tested antibiotic MIC values in B. anthracis. Collectively, these results confirm that WalRK responds to cell envelope stress in B. anthracis.

Comprehensive exploration unveiling the sonography and histopathology of uterine leiomyoma in a cow.

Genital tumours are rare among cattle, largely due to their relatively short lifespans. Leio-myoma, a smooth muscle tumour being more prevalent in dogs, appears only at a rate of 1.00 - 2.00% in cattle, affecting reproductive efficiency in cases of complete uterine obstruction. This case report involves an 8-year-old cow with repeated insemination attempts unveiled 5.00 cm intra-luminal uterine mass, obstructing the right uterine horn. Transrectal sonography (TRUS) revealed a highly vascularized mass with normal ovarian function. Confirmation of clinical condition, i.e., uterine leiomyoma, via uterine biopsy concluded the presence of neoplastic smooth muscle cells arranged in interlacing bundles showing mild pleomorphism, and special staining using Masson's trichrome revealed an unappreciable amount of connective tissue; subsequently right flank celiotomy was performed to remove the benign tumour. Forty-five days after celiotomy, TRUS examination confirmed an unobstructed uterine horn, and bilateral oviduct patency was adjudged with 2.50% methylene blue. Following treatment for chronic endometritis, artificial insemination led to conception nearly 90 days post-procedure. The TRUS aids preliminary diagnosis, while definitive identification demands necropsy and surgical methods. This case underscores the diagnostic significance of TRUS, histopathology and celiotomy for identifying and managing uterine leiomyoma in cattle.

KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.