The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

The Calpain System

Several well-recognized puzzles in microbiology have remained unsolved for decades. These include latent bacterial infections, unculturable microorganisms, persister cells and biofilm multidrug tolerance. Accumulating evidence suggests that these seemingly disparate phenomena result from the ability of bacteria to enter into a dormant (non-dividing) state. The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.

http://www.medicinabiomolecular.com.br/biblioteca/pdfs/Cancer/ca-3102.pdf

Persister cells, dormancy and infectious disease

CD95 (APO-1/Fas) is a prototype death receptor characterized by the presence of an 80 amino acid death domain in its cytoplasmic tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-CD95 antibodies or cognate CD95 ligand that initiate apoptosis. The complex of proteins that forms upon triggering of CD95 is called the death-inducting signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. This review discusses recent developments in this area of death receptor research.

/pdf/the-cd95-apo-1-fas-disc-and-beyond-22biv5u8xx.pdf

The CD95(APO-1/Fas) DISC and beyond

The genome DNA of Escherichia coli is associated with about 10 DNA-binding structural proteins, altogether forming the nucleoid. The nucleoid proteins play some functional roles, besides their structural roles, in the global regulation of such essential DNA functions as replication, recombination, and transcription. Using a quantitative Western blot method, we have performed for the first time a systematic determination of the intracellular concentrations of 12 species of the nucleoid protein in E. coli W3110, including CbpA (curved DNA-binding protein A), CbpB (curved DNA-binding protein B, also known as Rob [right origin binding protein]), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Qβ), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td mutant phenotype A). Intracellular protein levels reach a maximum at the growing phase for nine proteins, CbpB (Rob), DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp, and StpA, which may play regulatory roles in DNA replication and/or transcription of the growth-related genes. In descending order, the level of accumulation, calculated in monomers, in growing E. coli cells is Fis, Hfq, HU, StpA, H-NS, IHF*, CbpB (Rob), Dps*, Lrp, DnaA, IciA, and CbpA* (stars represent the stationary-phase proteins). The order of abundance, in descending order, in the early stationary phase is Dps*, IHF*, HU, Hfq, H-NS, StpA, CbpB (Rob), DnaA, Lrp, IciA, CbpA, and Fis, while that in the late stationary phase is Dps*, IHF*, Hfq, HU, CbpA*, StpA, H-NS, CbpB (Rob), DnaA, Lrp, IciA, and Fis. Thus, the major protein components of the nucleoid change from Fis and HU in the growing phase to Dps in the stationary phase. The curved DNA-binding protein, CbpA, appears only in the late stationary phase. These changes in the composition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the genome functions.

Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid

Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanisms of MDT and the nature of persisters have remained elusive. Our previous research has shown that persisters are largely responsible for the recalcitrance of biofilm infections. A general method for isolating persisters was developed, based on lysis of regular cells by ampicillin. A gene expression profile of persisters contained toxin-antitoxin (TA) modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function (for example, aminoglycosides interrupt translation, producing toxic peptides). We reasoned that inhibition of translation will lead to a shutdown of cellular functions, preventing antibiotics from corrupting their targets, giving rise to MDT persister cells. Overproduction of the RelE toxin, an inhibitor of translation, caused a sharp increase in persisters. Functional expression of a putative HipA toxin also increased persisters, while deletion of the hipBA module caused a sharp decrease in persisters in both stationary and biofilm populations. HipA is thus the first validated persister-MDT gene. We suggest that random fluctuation in the levels of MDT proteins leads to the formation of rare persister cells. The function of these specialized dormant cells is to ensure the survival of the population in the presence of lethal factors.

Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.

Proteomic Analysis of the Mammalian Mitochondrial Ribosome IDENTIFICATION OF PROTEIN COMPONENTS IN THE 28 S SMALL SUBUNIT

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli

Obg proteins belong to a subfamily of GTP binding proteins, which are highly conserved from bacteria to human. Mutations of obgE genes cause pleiotropic defects in various species but the function remained unclear. Here we examine the function of ObgE, the Obg homolog in Escherichia coli. The growth rate correlates with the amount of ObgE in cells. Co-fractionation experiments further suggest that ObgE binds to 30S and 50S ribosomal subunits, but not to 70S ribosome. Pull-down assays suggest that ObgE associates with several specific ribosomal proteins of 30S and 50S subunits, as well as RNA helicase CsdA. Purified ObgE cosediments with 16S and 23S ribosomal RNAs in vitro in the presence of GTP. Finally, mutation of ObgE affects pre-16Sr-RNA processing, ribosomal protein levels, and ribosomal protein modification, thereby significantly reducing 70S ribosome levels. This evidence implicates that ObgE functions in ribosomal biogenesis, presumably through the binding to rRNAs and/or rRNA-ribosomal protein complexes, perhaps as an rRNA/ribosomal protein folding chaperone or scaffold protein.

The GTP binding protein Obg homolog ObgE is involved in ribosome maturation

http://www.jbc.org/content/early/2001/02/21/jbc.M100432200.full.pdf

Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome. Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria.

Ribosomal modulation factor (RMF), a small basic protein, expresses transcriptionally in the stationary phase of Escherichia coli cells and binds to 50S ribosomal subunits. The RMF bound 70S ribosomes dimerize to form 100S particles that have no translational activity. In transferring the stationary cells to a fresh medium, the 100S particles release the RMF and dissociate to active 70S. The interconversion of ribosomes between active 70S and inactive 100S by RMF is a cellular mechanism controlling translation.

Akira Wada

Papers

Proteomic Analysis of the Mammalian Mitochondrial Ribosome IDENTIFICATION OF PROTEIN COMPONENTS IN THE 28 S SMALL SUBUNIT

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli

The GTP binding protein Obg homolog ObgE is involved in ribosome maturation

Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome. Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria.

Growth phase coupled modulation of Escherichia coli ribosomes.