Structural Investigation of Orf Virus Bcl-2 Homolog ORFV125 Interactions with BH3-Motifs from BH3-Only Proteins Puma and Hrk.

Numerous viruses have evolved sophisticated countermeasures to hijack the early programmed cell death of host cells in response to infection, including the use of proteins homologous in sequence or structure to Bcl-2. Orf virus, a member of the parapoxviridae, encodes for the Bcl-2 homolog ORFV125, a potent inhibitor of Bcl-2-mediated apoptosis in the host. ORFV125 acts by directly engaging host proapoptotic Bcl-2 proteins including Bak and Bax as well as the BH3-only proteins Hrk and Puma. Here, we determined the crystal structures of ORFV125 bound to the BH3 motif of proapoptotic proteins Puma and Hrk. The structures reveal that ORFV125 engages proapoptotic BH3 motif peptides using the canonical ligand binding groove. An Arg located in the structurally equivalent BH1 region of ORFV125 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that mimics the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. These findings provide a structural basis for Orf virus-mediated inhibition of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways.

Design, synthesis and biological evaluation of 3-aryl-rhodanine benzoic acids as anti-apoptotic protein Bcl-2 inhibitors.

A new class of 3-aryl-rhodanine benzoic acid derivatives were designed, synthesized, and evaluated for their inhibition activities against anti-apoptotic Bcl-2 proteins. The potent compounds 33 and 41 bound to Bcl-2 with submicromolar Ki values and had selectivities to Bcl-2/Mcl-1 over Bcl-xL. In addition, they exhibited obvious antiproliferative activities in three human tumor cell lines (MDA-MB-231, K562 and PC-3).

Functions of the C-terminal domains of apoptosis-related proteins of the Bcl-2 family.

Bcl-2 family proteins are involved in cell homeostasis, where they regulate cell death. Some of these proteins are pro-apoptotic and others pro-survival. Moreover, many of them share a similar domain composition with several of the so-called BH domains, although some only have a BH3 domain. A C-terminal domain is present in all the multi-BH domain proteins and in some of the BH3-only ones. This C-terminal domain is hydrophobic or amphipathic, for which reason it was thought when they were discovered that they were membrane anchors. Although this is indeed one of their functions, it has since been observed that they may also serve as regulators of the function of some members of this family, such as Bax. They may also serve to recognize the target membrane of some of these proteins, which only after an apoptotic signal, are incorporated into a membrane. It has been shown that peptides that imitate the sequence of C-terminal domains can form pores and may serve as a model to design cytotoxic molecules.

Dendritic cells in neointima formation after rat carotid balloon injury: coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair.

OBJECTIVES: We sought to evaluate: 1) the contribution of dendritic cells (DCs); and 2) the impact of B-cell lymphoma 2 protein (Bcl-2), a central anti-apoptotic protooncogene, and of heat shock protein 47 (HSP47), indicating subsequent collagen deposition, in neointima formation after angioplasty. BACKGROUND: The origin of neointimal cells and the factors that promote their accumulation are still unclear. Previous studies reported intimal presence of DCs and suggested cells of primarily extravascular origin to contribute to arterial repair. METHODS: Sprague-Dawley rats underwent carotid balloon angioplasty. At different times after angioplasty, tissue sections were analyzed by immunohistochemistry using OX-62 and S100 as DC markers and antibodies against Bcl-2 and HSP47, supplemented by electron microscopic analysis of cell type and apoptosis. RESULTS: Four days after injury, DCs adhered along the internal elastic lamina and demonstrated intense Bcl-2 and HSP47 expression, consistent with low apoptosis. With ongoing neointima enlargement, luminal DCs remained prevalent and were colocalized with Bcl-2 and HSP47, while signaling decreased to basal regions. Media showed no DCs and only low Bcl-2 and HSP47 immunoreactivity. Adventitia transiently revealed a structural separation between day 4 and 7. Whereas the inner layer demonstrated sparse cellularity, apoptosis and no DC, Bcl-2, and HSP47 labeling, the outer layer was characterized by high myofibroblast density with strong Bcl-2 and HSP47 expression but absence of DCs. CONCLUSIONS: We identify DCs as novel components in early neointima formation, promoted by coordinated anti-apoptotic Bcl-2 and HSP47 expression. Despite intense adventitial remodeling, there is no evidence of adventitial cell transmigration.

Increased expression and redistribution of the antiapoptotic molecule Bcl-xL in Parkinson's disease.

In the present study, we tried to clarify the potentially protective role of Bcl-x(L), an anti-apoptotic member of the Bcl-2 family of proteins, in Parkinson's disease (PD). Using in situ hybridization on human postmortem mesencephalon sections, we show that in PD patients Bcl-x(L) mRNA expression per dopaminergic neuron was almost double that of controls. We also show that, ultrastructurally, this effect may be mediated by a redistribution of Bcl-x(L) from the cytosol to the outer mitochondrial membrane.

La transfección de la línea celular TF-1 con el proto-oncogen bcl-2 humano proporciona supervivencia a corto plazo en ausencia de GM-CSF sin cambiar el fenotipo / Transfection of the tf-1 cell line with the human protooncogen bcl-2 favors short term cell survival and does not change phenotype

La muerte celular programada (apoptosis) es un proceso controlado genéticamente. El producto del gen bcl-2 (Bcl-2) es una proteína anti apoptótica integrada a la membrana externa mitocondrial. Objetivo. El objetivo del presente trabajo fue valorar los efectos de la transfección de bcl-2 sobre la supervivencia, proliferación, fenotipo y morfología en las líneas celulares TF-1, TF-1 neo (control negativo) y TB-1 (TF-1 transfectada con bcl-2). Metodología. Para ello utilizamos el método siguiente: examen del nivel de expresión de los antígenos de superficie CD13, CD34 y c-Kit, respuesta celular al GM-CSF y capacidad de supervivencia luego del retiro del factor de viabilidad. La apoptosis fue evaluada mediante la observación del blebbing membranal a diferentes tiempos luego del retiro del suero, el factor de viabilidad GM-CSF y la tolerancia a toxinas presentes en Justicia spicigera. Resultados. La expresión ectópica de bcl-2 en las células TB-1 previno la muerte celular por apoptosis sin inducir un cambio importante en el fenotipo y morfología en ausencia de GM-CSF, suero o en presencia del extracto de Justicia spicigera. Consistente con la función de Bcl-2, encontramos que la proliferación de la línea celular TB-1 fue muy similar a la línea parental TF-1 en respuesta al GM-CSF y por lo tanto Bcl-2 no altera dicha función. El retiro de suero y GM-CSF al medio de cultivo de las líneas TF-1 y TF-1 neo, induce apoptosis en 36 h. Por el contrario, la línea TB-1 entra en apoptosis a 96 h bajo las mismas condiciones. Conclusiones. Reuniendo los resultados, éstos sugieren que Bcl-2 es un inhibidor de la apoptosis a corto plazo en la línea celular TB-1, no cambia la respuesta al GM-CSF y no afecta el nivel de expresión de otros antígenos de superficie como CD13, CD34 y c-Kit.

[Expression of proto-oncogene bcl-2 in squamous cell carcinoma of the larynx]. / Expresión del proto-oncogén bcl-2 en el carcinoma epidermoide de laringe.

Apoptosis, or programmed cell death, is responsible for deleting cells in normal tissues to maintain homeostasis after DNA damage. Apoptosis has several physiological inhibitors, one of the most important being the proto-oncogene bcl-2. An immunohistochemical study was made of bcl-2 expression in 25 squamous cell carcinomas of the larynx, in laryngeal mucosa distant from the primary neoplasm, and in lymph node metastases. The relationship between bcl-2 expression and various clinical and pathological parameters was investigated. Bcl-2 was detected in 40% of primary tumors and in 71% of lymph node metastases; it seems to be a late event in laryngeal malignant transformation. We found no statistical association between bcl-2 expression and most of the clinical and pathological parameters examined. Only tumor differentiation was related to bcl-2 expression, bcl-2 positive tumors being moderately or poorly differentiated (p < 0.02).