ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.     

Structure–function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins

Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air–liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein–protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.     

Restriction factors of retroviral replication: the example of Tripartite Motif (TRIM) protein 5α and 22

Viral tropism, replication, and pathogenesis are determined by multiple interactions between the pathogen and the host. In the case of retroviruses, and in particular, the human immunodeficiency virus, the specific interaction of the envelope protein with the host receptors and co-receptors is essential to gain entry in the cells. After entry, the success of retroviruses to complete their life cycle depends on a complex interplay between the virus and host proteins. Indeed, the cell environment is endowed with a number of factors that actively block distinct stage(s) in the microbial life cycle. Among these restriction factors, Tripartite Motif-5α (TRIM5α) has been extensively studied; however, other TRIM family members have been demonstrated to be anti-retroviral effector proteins. This article reviews, in particular, the current knowledge on the anti-retroviral effects of TRIM5α and TRIM22.     

Calcineurin ensures a link between the DNA replication checkpoint and microtubule-dependent polarized growth

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.     

Evolutionary conservation of the 3′ ends of members of a family of giant secretory protein genes inChironomus pallidivittatus

Summary Two cDNA clones representing the 3-end regions of BR1 and BR2 75S mRNA were obtained fromChironomus pallidivittatus. The regular structure characterizing the core of these genes, consisting of tandemly arranged repeat units, changes into a more irregular structure toward the 3 end. Distal to a standard type of repeat unit with a characteristic excess of positive charges, a new type of repeat with a high, negative charge density is interspersed among parts of the standard unit. The last 111 amino acids before the stop codon represent a unique region distinctly different in amino acid composition from upstream regions, and include two partially homologous hydrophobic regions. Sequence comparison of 3-end regions from clones representing BR1 and BR2 genes indicates striking sequence conservation in the unique part of the region. Analysis of the level of silent site divergence shows that the homology increases in the 3 direction up to the polyadenylation site. That the unique region is retained as a part of the secreted protein is shown by Western blotting.     

Functional analysis of the interaction between the mismatch repair protein MutS and the replication processivity factor β clamp in Pseudomonas aeruginosa

Interaction between MutS and the replication factor β clamp has been extensively studied in a Mismatch Repair context; however, its functional consequences are not well understood. We have analyzed the role of the MutS-β clamp interaction in Pseudomonas aeruginosa by characterizing a β clamp binding motif mutant, denominated MutSβ, which does not interact with the replication factor. A detailed characterization of P. aeruginosa strain PAO1 harboring a chromosomal mutSβ allele demonstrated that this mutant strain exhibited mutation rates to rifampicin and ciprofloxacin resistance comparable to that of the parental strain. mutSβ PAO1 was as proficient as the parental strain for DNA repair under highly mutagenic conditions imposed by the adenine base analog 2-aminopurine. In addition, using a tetracycline resistance reversion assay to assess the repair of a frameshift mutation, we determined that the parental and mutSβ strains exhibited similar reversion rates. Our results clearly indicate that the MutS-β clamp interaction does not have a central role in the methylation-independent Mismatch Repair of P. aeruginosa.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

What does the homology between E. coli tRNAs and RNAs controlling ColE1 plasmid replication mean?

Nucleotide sequences of E. coli tRNAs and RNA I or RNA II (controlling replication of ColE1 plasmids) were compared using the computer. The homology between some of these molecules is over 60%. The distribution of homologous nucleotides among the functional elements (stems and loops) of either RNA I or RNA II and the tRNAs molecules was studied. It was found that the homologous domains are located mainly in the loop regions of RNA I or RNA II. A consensus sequence, the nonanucleotide AGUUGGUAG, was discovered in loop II of RNA I and in the dihydrouridylic loop of tRNAs showing homology with RNA I. Based on this observation, a hypothesis was drawn for a possible role of the tRNAs in the regulation of plasmid DNA replication.     

Nicotine: the link between cigarette smoking and the progression of renal injury?

Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) alpha4, alpha5, alpha7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca(2+) channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 (-7) M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 +/- 50 vs. nicotine, 2,761 +/- 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 +/- 26 vs. nicotine, 281.5 +/- 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.     

Control of Dead end localization and activity – Implications for the function of the protein in antagonizing miRNA function

Dead end (dnd) is a vertebrate-specific component of the germ plasm and germ-cell granules that is crucial for germ-cell development in zebrafish and mouse. Dnd counteracts the inhibitory function of miRNAs, thereby facilitating the expression of proteins such as Nanos and Tdrd7 in the germ cells. Here, we show that cis-acting elements within dnd mRNA and the RNA recognition motive (RRM) of the protein are essential for targeting protein expression to the germ cells and to the perinuclear granules, respectively. We demonstrate that as it executes its function, Dnd translocates between the germ-cell nucleus and germ-cell granules. This phenomenon is not observed in proteins mutated in the RRM motif, correlating with loss of function of Dnd. Based on molecular modeling, we identify the putative RNA binding domain of Dnd as a canonical RRM and propose that this domain is important for protein subcellular localization and function.     

Smc5/6: a link between DNA repair and unidirectional replication?

Of the three structural maintenance of chromosome (SMC) complexes, two directly regulate chromosome dynamics. The third, Smc5/6, functions mainly in homologous recombination and in completing DNA replication. The literature suggests that Smc5/6 coordinates DNA repair, in part through post-translational modification of uncharacterized target proteins that can dictate their subcellular localization, and that Smc5/6 also functions to establish DNA-damage-dependent cohesion. A nucleolar-specific Smc5/6 function has been proposed because Smc5/6 yeast mutants display penetrant phenotypes of ribosomal DNA (rDNA) instability. rDNA repeats are replicated unidirectionally. Here, we propose that unidirectional replication, combined with global Smc5/6 functions, can explain the apparent rDNA specificity.     

Signaling factors of self-organization of protein synthesis rhythm in hepatocyte cultures—gangliosides and catecholamines function independently

Considering the data on the low level of self-organization (self-synchronization) of protein synthesis rhythm in aging, we studied the possible interference of the signaling factors of self-organization, gangliosides and catecholamines, as well as catecholamine reception. Experiments were carried out on primary cultures of rat hepatocytes on slides. Inhibited ganglioside synthesis did not prevent the organization of protein synthesis rhythm by the α-adrenomimetic agent phenylephrine. Upon the blockade of α-receptors by prazosin, the protein synthesis rhythm was observed after the exposure to gangliosides. α-Adrenolytic agents prazosin and benoxathian abolished the synchronizing effect of the β-adrenomimetic isoproterenol. A mixture of α-and β-adrenomimetic agents inhibited the protein synthesis rhythm-organizing effect of noradrenaline. Thus, the signaling molecules of self-organization of protein synthesis function independently via specific receptors.     

Evidence of homology between the β-chain of human haptoglobin and the chymotrypsin family of serine proteases

Amino acid sequences from the β-chain of human haptoglobin are compared with those sequences known for the serine proteases of the chymotrypsin family. In a comparison of some 171 residues of the haptoglobin β-chain (approximately 60% of the protein molecule), approximately 30% of these are identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsin A, bovine chymotrypsin B, porcine elastase, or bovine thrombin B-chain, and an additional 10% are chemically similar. A combined comparison of the haptoglobin β-chain with the above five serine proteases gave an identity of 56% and a chemical similarity of 11%. Similarity of primary structure is also striking around two of the five half-cystinyl residues so far characterized in long lengths of sequence. These data provide substantial evidence that the β-chain of haptoglobin is homologous to the chymotrypsin family of serine proteases. Proposals are also presented to explain the occurrence of internal homology in the N-terminal region of the β-chain.     

Evolutionary analysis of the multigene pregnancy-specific β1-Glycoprotein family: Separation of historical and nonhistorical signals

The pregnancy-specific ₁-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.