The human mitochondrial genome encodes 13 proteins, all subunits of the respiratory chain complexes and thus involved in energy metabolism. These genes are translated by 22 transfer RNAs (tRNAs), also encoded by the mitochondrial genome, which form the minimal set required for reading all codons. Human mitochondrial tRNAs gained interest with the rapid discovery of correlations between point mutations in their genes and various neuromuscular and neurodegenerative disorders. In this review, emerging fundamental knowledge on the structure/function relationships of these particular tRNAs and an overview of the large variety of mechanisms within translation, affected by mutations, are summarized. Also, initial results on wide-ranging molecular consequences of mutations outside the frame of mitochondrial translation are highlighted. While knowledge of mitochondrial tRNAs in both health and disease increases, deciphering the intricate network of events leading different genotypes to the variety of phenotypes requires further investigation using adapted model systems.

Human Spindly is required for kinetochore localization of cytoplasmic dynein, which is essential for poleward movement of chromosomes and for kinetochore protein streaming. In addition, Spindly controls the activity and kinetochore abundance of the RZZ complex, which contributes to microtubule attachment and mitotic checkpoint activity.

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.

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Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a 'late-stage' event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half -those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond. Cell Death and Differentiation (2009) 16, 195-207; doi:10.1038/cdd.2008 published online 21 November 2008 On the basis of structural and functional characteristics, caspases involved in apoptosis are grouped into 'initiator' caspases, containing a long prodomain, such as the death effector domain found in mammalian caspases-8 and -10 or the caspase activation and recruitment domain (CARD) found in caspases-2 and -9, as well as short prodomain containing effector caspases-3, -6 and -7. Initiator caspases are activated by autocatalytic processing, which is initiated upon prodomain-dependent dimerization of zymogens at specific activation platforms such as the 'apoptosome' or the 'deathinducing signaling complex' (DISC).1 The number of substrates cleaved by initiator caspases is thought to be limited to themselves and downstream effector caspases. The BH3-only Bcl-2 family member Bid appears to be the only exception, as it can also be cleaved by initiator caspases, to connect the extrinsic apoptotic pathway with the mitochondrial apoptosis signaling pathway. 2,3A molecule at odds with current dogma is caspase-2 (earlier synonyms Ich-1: ICE/CED-3 homolog 1 or Nedd-2, for neural precursor cells-expressed, developmentally downregulated), the most conserved caspase across species, sharing 55% similarity with Caenorhabditis elegans caspase Ced-3.4-6 On the one hand, the predicted cleavage specificity of caspase-2 appears to place it more closely to effector caspases-3 and -7. 7 On the other, caspase-2 contains a long CARD prodomain, through which it can interact with adaptor proteins, which is typical for initiator caspases. Overall, biochemical analysis supports a role of caspase-2 as an initiator caspase, but many contradictory findings render the exact function of this enzyme unresolved. In this review, we aim to give an overview on the steadily growing body of literature about this enigmatic enzyme, aiming to provide a synopsis on the present knowledge and to point out important aspects that still need to be tackled in future research. Activation and Processing of Caspase-2During apoptosis induction, caspase-2 is thought to be activated by CARD-mediated, dimerization-induced intrasubunit cleavage (@D333) and subsequent removal of the prodomain (@D169) as well as of the linker region (@D347), connecting its large p19 subunit with the sm...

Large-scale production and incorporation of titanium dioxide nanoparticles (NP-TiO2 ) in consumer products leads to their potential release into the environment and raises the question of their toxicity. The bactericidal mechanism of NP-TiO2 under UV light is known to involve oxidative stress due to the generation of reactive oxygen species. In the dark, several studies revealed that NP-TiO2 can exert toxicological effects. However, the mode of action of these nanoparticles is still controversial. In the present study, we used a combination of fluorescent probes to show that NP-TiO2 causes Escherichia coli membrane depolarization and loss of integrity, leading to higher cell permeability. Using both transcriptomic and proteomic global approaches we showed that this phenomenon translates into a cellular response to osmotic stress, metabolism of cell envelope components and uptake/metabolism of endogenous and exogenous compounds. This primary mechanism of bacterial NP-TiO2 toxicity is supported by the observed massive cell leakage of K(+) /Mg(2+) concomitant with the entrance of extracellular Na(+), and by the depletion of intracellular ATP level.

Mutations in the rfa operon leading to severely truncated lipopolysaccharide (LPS) structures are associated with pleiotropic effects on bacterial cells, which in turn generates a complex phenotype termed deep-rough. Literature reports distinct behavior of these mutants in terms of susceptibility to bacteriophages and to several antibacterial substances. There is so far a critical lack of understanding of such peculiar structure-reactivity relationships mainly due to a paucity of thorough biophysical and biochemical characterizations of the surfaces of these mutants. In the current study, the biophysicochemical features of the envelopes of Escherichia coli deep-rough mutants are identified from the molecular to the single cell and population levels using a suite of complementary techniques, namely microelectrophoresis, Atomic Force Microscopy (AFM) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) for quantitative proteomics. Electrokinetic, nanomechanical and proteomic analyses evidence enhanced mutant membrane destabilization/permeability, and differentiated abundances of outer membrane proteins involved in the susceptibility phenotypes of LPS-truncated mutants towards bacteriophages, antimicrobial peptides and hydrophobic antibiotics. In particular, inner-core LPS altered mutants exhibit the most pronounced heterogeneity in the spatial distribution of their Young modulus and stiffness, which is symptomatic of deep damages on cell envelope likely to mediate phage infection process and antibiotic action.

Tight transcriptional regulation, post-translational modifications and/or alternative splicing of BH3-only proteins fine-tune their pro-apoptotic function. Here, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2 modifying factor) and describe the generation of two major isoforms from a common transcript where initiation of protein synthesis involves leucine-coding CUG. BmfCUG and the originally described isoform, Bmf short (BmfS), display comparable binding affinities to pro-survival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced upon forms of cell stress known to cause the repression of the CAP-dependent translation machinery such as serum-deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of eukaryotic translation initiation factor eIF-4E. Knock-down or deletion of Bmf reduces apoptosis under some of these conditions demonstrating that Bmf can act as a sentinel for the stress-impaired CAP-dependent protein translation machinery (150).