Role of C-terminal domain of Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial stress and macrophage apoptosis.

PE/PPE proteins of Mycobacterium tuberculosis (Mtb) target the host organelles to dictate the outcome of infection. This study investigated the significance of PE6/Rv0335c protein's unique C-terminal in causing host mitochondrial perturbations and apoptosis. In-silico analysis revealed that similar to eukaryotic apoptotic Bcl2 proteins, Rv0335c had disordered, hydrophobic C-terminal and two BH3-like motifs in which one was located at C-terminal. Also, Rv0335c's N terminal had mitochondrial targeting sequence. Since, C-terminal of Bcl2 proteins are crucial for mitochondria targeting and apoptosis; it became relevant to evaluate the role of Rv0335c's C-terminal domain in modulating host mitochondrial functions and apoptosis. To confirm this, in-vitro experiments were conducted with Rv0335c whole protein and Rv0335c∆Cterm (C-terminal domain deleted Rv0335c) protein. Rv0335c∆Cterm caused significant reduction in mitochondrial perturbations and Caspase-mediated apoptosis of THP1 macrophages in comparison to Rv0335c. However, the deletion of C-terminal domain didn't affect Rv0335c's ability to localize to mitochondria. Nine Ca2+ binding residues were predicted within Rv0335c and four of them were at the C-terminal. In-vitro studies confirmed that Rv0335c caused significant increase in intracellular calcium influx whereas Rv0335c∆Cterm had insignificant effect on Ca2+ influx. Rv0335c has been reported to be a TLR4 agonist and, we observed a significant reduction in the expression of TLR4-HLA-DR-TNF-α in response to Rv0335c∆Cterm protein also suggesting the role of Rv0335c's C-terminal domain in host-pathogen interaction. These findings indicate the possibility of Rv0335c as a molecular mimic of eukaryotic Bcl2 proteins which equips it to cause host mitochondrial perturbations and apoptosis that may facilitate pathogen persistence.

NMR characterization of the interaction between Bcl-xL and the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 µM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 µM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

Induction of apoptosis in colorectal cancer cells by matrix protein of PPR virus as a novel anti-cancer agent.

Colorectal cancer (CRC) is a common and highly malignant neoplasm, ranking as the fourth most frequent cause of cancer-related deaths worldwide. Recently, non-human oncolytic viruses such as Peste des petits ruminants virus (PPRV) are considered as a potent candidate in the viral therapy of cancer. In the current study, the apoptotic effects of matrix (M) protein of PPRV was investigated on SW480 CRC cells. The M gene was cloned into the pcDNA™3.1/Hygro(+) expression vector and transfected into the cancer cells. The cytotoxic effects of the M protein on SW480 cells were confirmed using MTT assay. Furthermore, flow cytometry results showed that the M protein induces apoptosis in 91 % of CRC cells. Interestingly, the expression of the M gene in SW480 cells led to the up-regulation of genes including Bax, p53, and Caspase-9, as well as an increase in the Bax/Bcl-2 ratio. By using bioinformatics modeling, we hypothesized that the M protein could interact with Bax factor through its BH3-like motif and could further activate the intrinsic apoptosis pathway. Ultimately, this study provided the first evidence of the pro-apoptotic activity of PPRV M protein indicating its possible development as a promising novel anti-cancer agent.

Chemical shift assignments of a fusion protein comprising the C-terminal-deleted hepatitis B virus X protein BH3-like motif peptide and Bcl-xL.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV has the multifunctional protein, HBV X protein (HBx, 154 residues), which plays key roles in HBV replication and liver disease development. Interaction of HBx through its BH3-like motif with the anti-apoptotic protein Bcl-xL leads to HBV replication and induction of apoptosis, resulting in HCC development. Our previous nuclear magnetic resonance (NMR) study revealed that the HBx BH3-like motif peptide (residues 101-136) binds to the common BH3-binding groove of Bcl-xL. Importantly, a C-terminal-truncated HBx, e.g., residues 1-120 of HBx, is strongly associated with the increased risk of HBV-related HCC development. However, the interaction mode between the C-terminal-truncated HBx and Bcl-xL remains unclear. To elucidate this interaction mode, the C-terminal-deleted HBx BH3-like motif peptide (residues 101-120) was used as a model peptide in this study. To facilitate the NMR analysis, we prepared a fusion protein of HBx (101-120) and Bcl-xL connected with five repeats of the glycine-serine dipeptide as a linker. Here, we report the 1H, 13C, and 15N resonance assignments of the fusion protein. This is the first step for the elucidation of the pathogenesis of liver diseases caused by the interaction between the C-terminal-truncated HBx and Bcl-xL.

Telomerase Reverse Transcriptase Contains a BH3-Like Motif and Interacts with BCL-2 Family Members.

Upregulation of human telomerase reverse transcriptase (hTERT) expression is an important factor in the cellular survival and cancer. Although growing evidence suggests that hTERT inhibits cellular apoptosis by telomere-independent functions, the mechanisms involved are not fully understood. Here, we show that hTERT contains a BH3-like motif, a short peptide sequence found in BCL-2 family proteins, and interacts with anti-apoptotic BCL-2 family proteins MCL-1 and BCL-xL, suggesting a functional link between hTERT and the mitochondrial pathway of apoptosis. Additionally, we propose that hTERT can be categorized into the atypical BH3-only proteins that promote cellular survival, possibly due to the non-canonical interaction between hTERT and antiapoptotic proteins. Although the detailed mechanisms underlying the hTERT BH3-like motif functions and interactions between hTERT and BCL-2 family proteins have not been elucidated, this work proposes a possible connection between hTERT and BCL-2 family members and reconsiders the role of the BH3-like motif as an interaction motif.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies.

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.