Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression

Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Delta null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.     

Asc1p,a ribosomal protein,plays a pivotal role in cellular adhesion and virulence in <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans, the common human fungal pathogen, can switch morphology from yeast to pseudohyphal or hyphal form upon various environmental cues. It is well-known that the ability of morphological conversion and adhesive growth renders C. albicans virulent. It is noteworthy that every factor involved in the morphogenesis is known to be important for the virulence of this pathogen. To examine a functional relevance of Asc1p, a ribosomal protein, in morphogenesis and virulence, an asc1 homozygous null mutant was generated. Although a normal morphological transition of the asc1 deletion strain in liquid media was found, it did not change its morphology on solid media. Moreover, the adhesion activity and hyphal-specific gene expression were defective due to ASC1 deletion. Finally, it was found that the asc1 null mutant was avirulent in a mouse model. These results strongly suggested that Asc1p a component of the 40S ribosomal subunit and a signal transducer, plays a pivotal role in cellular adhesion and virulence through regulation of specific gene expression in C. albicans.     

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.     

Identification of a novel response regulator, Crr1, that is required for hydrogen peroxide resistance in Candida albicans

Candida albicans colonises numerous niches within humans and thus its success as a pathogen is dependent on its ability to adapt to diverse growth environments within the host. Two component signal transduction is a common mechanism by which bacteria respond to environmental stimuli and, although less common, two component-related pathways have also been characterised in fungi. Here we report the identification and characterisation of a novel two component response regulator protein in C. albicans which we have named CRR1 (Candida Response Regulator 1). Crr1 contains a receiver domain characteristic of response regulator proteins, including the conserved aspartate that receives phosphate from an upstream histidine kinase. Significantly, orthologues of CRR1 are present only in fungi belonging to the Candida CTG clade. Deletion of the C. albicans CRR1 gene, or mutation of the predicted phospho-aspartate, causes increased sensitivity of cells to the oxidising agent hydrogen peroxide. Crr1 is present in both the cytoplasm and nucleus, and this localisation is unaffected by oxidative stress or mutation of the predicted phospho-aspartate. Furthermore, unlike the Ssk1 response regulator, Crr1 is not required for the hydrogen peroxide-induced activation of the Hog1 stress-activated protein kinase pathway, or for the virulence of C. albicans in a mouse model of systemic disease. Taken together, our data suggest that Crr1, a novel response regulator restricted to the Candida CTG clade, regulates the response of C. albicans cells to hydrogen peroxide in a Hog1-independent manner that requires the function of the conserved phospho-aspartate.     

The transcriptional regulator Hap1p (Cyp1p) is essential for anaerobic or heme-deficient growth of Saccharomyces cerevisiae: Genetic and molecular characterization of an extragenic suppressor that encodes a WD repeat protein.

We report here that Hap1p (originally named Cyp1p) has an essential function in anaerobic or heme-deficient growth. Analysis of intragenic revertants shows that this function depends on the amino acid preceding the first cysteine residue of the DNA-binding domain of Hap1p. Selection of recessive extragenic suppressors of a hap1-hem1- strain allowed the identification, cloning, and molecular analysis of ASC1 (Cyp1 Absence of growth Supressor). The sequence of ASC1 reveals that its ORF is interrupted by an intron that shelters the U24 snoRNA. Deletion of the intron, inactivation of the ORF, and molecular localization of the mutations show unambiguously that it is the protein and not the snoRNA that is involved in the suppressor phenotype. ASC1, which is constitutively transcribed, encodes an abundant, cytoplasmically localized 35-kD protein that belongs to the WD repeat family, which is found in a large variety of eucaryotic organisms. Polysome profile analysis supports the involvement of this protein in translation. We propose that the absence of functional Asc1p allows the growth of hap1-hem1- cells by reducing the efficiency of translation. Based on sequence comparisons, we discuss the possibility that the protein intervenes in a kinase-dependent signal transduction pathway involved in this last function.     

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

Quality control systems monitor and stop translation at some ribosomal stalls, but it is unknown if halting translation at such stalls actually prevents synthesis of abnormal polypeptides. In yeast, ribosome stalling occurs at Arg CGA codon repeats, with even two consecutive CGA codons able to reduce translation by up to 50%. The conserved eukaryotic Asc1 protein limits translation through internal Arg CGA codon repeats. We show that, in the absence of Asc1 protein, ribosomes continue translating at CGA codons, but undergo substantial frameshifting with dramatically higher levels of frameshifting occurring with additional repeats of CGA codons. Frameshifting depends upon the slow or inefficient decoding of these codons, since frameshifting is suppressed by increased expression of the native tRNA^Arg(ICG) that decodes CGA codons by wobble decoding. Moreover, the extent of frameshifting is modulated by the position of the CGA codon repeat relative to the translation start site. Thus, translation fidelity depends upon Asc1-mediated quality control.     

Identification of an exoribonuclease homolog, CaKEM1/CaXRN1, in Candida albicans and its characterization in filamentous growth

KEM1/XRN1 and RAT1 are two known exoribonuclease genes in Saccharomyces cereivsiae and encode a cytoplasmic and nuclear exoribonuclease, respectively. CaKEM1/CaXRN1 and CaRAT1, the Candida albicans homologs of 5'-->3' exoribonuclease genes, were identified by protein sequence comparisons and by functional complementation of the S. cerevisiae kem1/xrn1 null mutation. The deduced amino acid sequences of CaKEM1 and CaRAT1 show 51% and 55% identities to those of the S. cerevisiae KEM1 and RAT1, respectively. The exonuclease motifs were found to be highly conserved in CaKem1p and CaRat1p. We disrupted two chromosomal copies of CaKEM1 in a diploid C. albicans strain and demonstrate that C. albicans kem1/kem1 mutants are defective in filamentous growth on filamentous-inducing media. These results imply that CaKEM1 is involved in filamentous growth of C. albicans.     

Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.     

Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.     

Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans

The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.     

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.     

Activation of inflammasomes in podocyte injury of mice on the high fat diet: Effects of ASC gene deletion and silencing

Inflammasome, an intracellular inflammatory machinery, has been reported to be involved in a variety of chronic degenerative diseases such as atherosclerosis, autoinflammatory diseases and Alzheimer's disease. The present study hypothesized that the formation and activation of inflammasomes associated with apoptosis associated speck-like protein (ASC) are an important initiating mechanism resulting in obesity-associated podocyte injury and consequent glomerular sclerosis. To test this hypothesis, Asc gene knockout (Asc^−/−), wild type (Asc^+/+) and intrarenal Asc shRNA-transfected wild type (Asc shRNA) mice were fed a high fat diet (HFD) or normal diet (ND) for 12 weeks to produce obesity and associated glomerular injury. Western blot and RT-PCR analyses demonstrated that renal tissue Asc expression was lacking in Asc^−/− mice or substantially reduced in Asc shRNA transfected mice compared to Asc^+/+ mice. Confocal microscopic and co-immunoprecipitation analysis showed that the HFD enhanced the formation of inflammasome associated with Asc in podocytes as shown by colocalization of Asc with Nod-like receptor protein 3 (Nalp3). This inflammasome complex aggregation was not observed in Asc^−/− and local Asc shRNA-transfected mice. The caspase-1 activity, IL-1β production and glomerular damage index (GDI) were also significantly attenuated in Asc^−/− and Asc shRNA-transfected mice fed the HFD. This decreased GDI in Asc^−/− and Asc shRNA transfected mice on the HFD was accompanied by attenuated proteinuria, albuminuria, foot process effacement of podocytes and loss of podocyte slit diaphragm molecules. In conclusion, activation and formation of inflammasomes in podocytes are importantly implicated in the development of obesity-associated glomerular injury.     

Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate

To search a regimen for prevention of post-ischemic reperfusional (I/R) injuries, I/R in the liver was induced by 30-min clamping and subsequent unfastening of the portal vein of a rat, which underwent previous intravenous administration with ascorbic acid (Asc) of 1 mg/kg or the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated Asc (Asc2G) or 2-O-phosphorylated Asc (Asc2P) of 1 mg Asc equivalent/kg from the viewpoint of utilization of antioxidants that can promptly scavenge I/R-derived reactive oxygen species. The administration with Asc, Asc2P or Asc2G prevented some features of hepatic I/R injuries such as release of hepatic marker enzymes GOT and GPT into the blood vessel, cellular degenerative symptoms including vacuolation and cell fragmentation, and nuclear DNA strand cleavage as detected by TUNEL staining. The preventive effects on I/R injuries were in the order: Asc2G > Asc2P >> Asc. This order of preventive degrees of three anti-oxidants is partly attributable to proper efficiency of conversion to vitamin C and stability in blood stream; Asc2P was moderately converted to a free monoanion form of Asc in human serum, but, in rat serum, so efficiently converted to Asc as to undergo the resultant oxidative decomposition before reaching the liver, whereas Asc2G underwent scarce conversion to Asc in human serum but moderate conversion in rat serum, suggesting that Asc2P might be less cytoprotective against I/R injury than Asc2G in the rat liver in a way different from the human liver. In contrast Asc was so susceptible to autooxidation as to be rapidly decomposed in either rat or human serum. The concentrations of ascorbyl radicals (AscR) in serum were unchanged during I/R for sham-operated rats, but appreciably diminished time-dependently for I/R-operated rats as shown by ESR spectra. A marked increase in serum AscR occurred in rats receiving Asc, Asc2G or Asc2P, but it was time-dependently restored down to the pre-ischemic level of AscR in I/R-operated rats more rapidly than in sham-operated rats. Thus, hepatic I/R injuries were shown to be prevented more markedly by Asc2G or Asc2P than by Asc, which is attributable to efficiencies of both vitamin C conversion and subsequent AscR retention.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.