Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius

Certain non-capsulate strains belonging to the Haemophilus influenzae/Haemophilus aegyptius complex show unusually high pathogenicity, but the evolutionary origin of these virulent phenotypes, termed H. influenzae biogroup aegyptius, is as yet unknown. The aim of the present study was to elucidate the mechanisms of evolution of two paralogous genes, hap and iga, which encode the adhesion and penetration Hap protein and the IgA1 protease respectively. Partial sequencing of hap and iga genes in a comprehensive collection of strains belonging to the H. influenzae/H. aegyptius complex revealed considerable genetic polymorphism and pronounced mosaic-like patterns in both genes, but no evidence of intrastrain recombination between the two genes. A conserved hap pseudogene was present in all strains of H. aegyptius and H. influenzae biogroup aegyptius, each of which constituted distinct subpopulations as revealed by phylogenetic analysis. There was no evidence for a second, functional copy of the hap gene in these strains. The perturbed expression of the Hap serine protease appears to be associated with the formation of elongated bacterial cells growing in chains and a distinct colonization pattern on conjunctival cells, previously termed microcolony formation. The fact that individual hap pseudogenes differed from the ancestral sequence by zero to two positions within a 1.5 kb stretch suggests that the silencing event happened approximately 2000-11,000 years ago. Divergence of H. aegyptius and H. influenzae biogroup aegyptius occurred subsequent to this genetic event. The loss of Hap protein expression may be one of the genetic events that facilitated exploitation of the conjunctivae as a new niche.     

Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70

Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.     

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1-Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1β, cathelicidin human cationic antimicrobial protein-18/LL-37 and β-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1β through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.     

Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants. The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.     

ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway

The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein-protein interaction domain. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53-Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53-Bax network.     

Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non-dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22-71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49-71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60-76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49-65 of p21Sdi1 were substituted with amino acids 60-76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.