Abdel Aouacheria

Evolution of Bcl-2 homology motifs: homology versus homoplasy

Bcl-2 family proteins regulate apoptosis in animals. This protein family includes several homologous proteins and a collection of other proteins lacking sequence similarity except for a Bcl-2 homology (BH)3 motif. Thus, membership in the Bcl-2 family requires only one of the four BH motifs. On this basis, a growing number of diverse BH3-only proteins are being reported. Although compelling cell biological and biophysical evidence validates many BH3-only proteins, claims of significant BH3 sequence similarity are often unfounded. Computational and phylogenetic analyses suggest that only some BH3 motifs arose by divergent evolution from a common ancestor (homology), whereas others arose by convergent evolution or random coincidence (homoplasy), challenging current assumptions about which proteins constitute the extended Bcl-2 family.

Bcl-wav and the mitochondrial calcium uniporter drive gastrula morphogenesis in zebrafish

Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT–PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

In silico whole-genome screening for cancer-related single-nucleotide polymorphisms located in human mRNA untranslated regions

BackgroundA promising application of the huge amounts of genetic data currently available lies in developing a better understanding of complex diseases, such as cancer. Analysis of publicly available databases can help identify potential candidates for genes or mutations specifically related to the cancer phenotype. In spite of their huge potential to affect gene function, no systematic attention has been paid so far to the changes that occur in untranslated regions of mRNA.ResultsIn this study, we used Expressed Sequence Tag (EST) databases as a source for cancer-related sequence polymorphism discovery at the whole-genome level. Using a novel computational procedure, we focused on the identification of untranslated region (UTR)-localized non-coding Single Nucleotide Polymorphisms (UTR-SNPs) significantly associated with the tumoral state. To explore possible relationships between genetic mutation and phenotypic variation, bioinformatic tools were used to predict the potential impact of cancer-associated UTR-SNPs on mRNA secondary structure and UTR regulatory elements. We provide a comprehensive and unbiased description of cancer-associated UTR-SNPs that may be useful to define genotypic markers or to propose polymorphisms that can act to alter gene expression levels. Our results suggest that a fraction of cancer-associated UTR-SNPs may have functional consequences on mRNA stability and/or expression.ConclusionWe have undertaken a comprehensive effort to identify cancer-associated polymorphisms in untranslated regions of mRNA and to characterize putative functional UTR-SNPs. Alteration of translational control can change the expression of genes in tumor cells, causing an increase or decrease in the concentration of specific proteins. Through the description of testable candidates and the experimental validation of a number of UTR-SNPs discovered on the secreted protein acidic and rich in cysteine (SPARC) gene, this report illustrates the utility of a cross-talk between in silico transcriptomics and cancer genetics.

In silico whole-genome scanning of cancer-associated nonsynonymous SNPs and molecular characterization of a dynein light chain tumour variant

Last decade has led to the accumulation of large amounts of data on cancer genetics, opening an unprecedented access to the mapping of cancer genes in the human genome. Single-nucleotide polymorphisms (SNPs), the most common form of DNA variation in humans, emerge as an invaluable tool for cancer association studies. These genotypic markers can be used to assay how alleles of candidate genes correlate with the malignant phenotype, and may provide new clues into the genetic modifications that characterize cancer onset. In this cancer-oriented study, we detail an SNP mining strategy based on the analysis of expressed sequence tags among publicly available databases. Our whole-genome approach provides a comprehensive and unbiased description of nonsynonymous SNPs (nsSNPs) in tumoral versus normal tissues. To gain further insights into the possible relationships between genetic variation and altered phenotype, locations of a subset of nsSNPs were mapped onto protein domains known to be critical for protein function. Computational methods were also used to predict the potential impact of these cancer-associated nsSNPs on protein structure and function. We illustrate our approach through the detailed biochemical and structural characterization of a previously unknown cancer-associated mutation (G79C) affecting the 8 kDa dynein light chain (DNCL1).

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

&NA; The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl‐2 protein family. Connecting mitochondrial dynamics with life‐death pathway forks may arise from the intersection of Bcl‐2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl‐2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl‐2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL‐2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl‐2 family proteins integrate into some of these processes. HighlightsMultiple features of mitochondrial morphology.Strategies for deducing mitochondrial dynamics from morphological features.Eliminating neuronal mitochondria by diverse strategies.Bcl‐2 family proteins influence mitochondrial dynamics in neurons.Technologies for quantifying mitochondrial dynamics.

Redefining the BH3 Death Domain as a 'Short Linear Motif'.

B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein–protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.

Modulating Mitochondria-Mediated Apoptotic Cell Death through Targeting of Bcl-2 Family Proteins

Research demonstrated that the function of mitochondria extends well beyond that of being cell powerhouses and revealed that these organelles fulfil a dual role in both cellular life and death. In most vertebrates, execution of the mitochondrial pathway of apoptosis requires permeabilization of the mitochondrial outer membrane, an event which allows for the release of a variety of intramembrane space proteins, leading to the activation of caspases and ultimately cell demise. Bcl-2 family proteins, which include pro- and antiapoptotic members, positively or negatively regulate mitochondrial outer membrane permeabilization, i.e. a barrier to apoptosis induction. Over-expression of Bcl-2 and Bcl-x(L) is associated with tumor progression and may be responsible for drug resistance, making pro-survival Bcl-2 family members important targets for the development of anticancer agents. Pharmacological apoptosis modulation by manipulation of pro-apoptotic Bcl-2 family proteins, with the goal to treat disorders associated with uncontrolled cell death or to kill unwanted cells, is likely to represent an additional research focus in the coming years. The purpose of this review is to describe, with examples taken from recent patents, novel strategies for targeting the Bcl-2 family of apoptotic regulators through peptide-based approaches and selective delivery of functional nucleic acids.

µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

p60(v-src) and serum control cell shape and apoptosis via distinct pathways in quail neuroretina cells.

We made use of QNR cells transformed by a thermosensitive (tsNY68) strain of the Rous sarcoma virus (RSV) to compare the effect of p60v-src and serum in cultured nerve cells. In this system, both p60v-src heat inactivation and serum removal resulted in growth arrest in G1. In both cases, growth arrest was reversible since cell proliferation was rapidly re-induced following respectively p60v-src renaturation or serum re-addition. However, cells did not fully recover their ability to grow in soft agar, suggesting that, in contrast to the cell cycle machinery, the transforming capacities of these cells have been irreversibly altered. We found that p60v-src kinase activity prevented detachment from the substratum and cell death following serum removal. Thermal inactivation of p60v-src at restrictive temperature (41.5°C), but not serum removal, resulted in dramatic morphological changes, which occured 4 h after temperature shift up to 41.5°C. Later on, typical features of apoptotic cells could be observed. Cell death was greatly reduced by the caspase-3 inhibitor ZVAD.FMK, but not by the caspase-1 inhibitor Ac-YVAD.CHO. Together, these results suggested that p60v-src and serum factors act on distinct pathways, at least in part. In an attempt to identify the signalling pathways involved in the cell response to p60v-src down regulation, we found that Erk and Rac were rapidly inactivated following temperature shift up to 41.5°C. Thus, the combined effects of p60v-src and serum factors on the cytoskeleton dynamics and the apoptosis machinery are essential for full neoplastic transformation of neuroretina cells.