The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.     

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.     

Molecular cloning of Saccharomyces cerevisiae MGC1/YDR473c gene which is essential for cell growth

Saccharomyces cerevisiae haploid cells undergo morphological changes in response to mating pheromones, a- and -factors, during sexual conjugation. As a first step to elucidate the mechanism, I had previously identified the mgc1 mutation which affected the morphogenesis induced by mating pheromones. The mutation had been designated mgc1 for morphogenesis control. In the present study I cloned the MGC1 gene. Sequencing analysis indicates that the MGC1 gene corresponds to the YDR473c gene. The MGC1 gene was shown to be essential for cell growth and required for the transition from the G1 to S phase of cell cycle. Protein-protein interaction of Mgc1 protein was shown by using yeast two-hybrid system. Mgc1 protein was also proposed to be localized in the nucleus in yeast cells.     

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

Mutations of the genes encoding aminoacyl-t RNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5 AG, p.D2 G; c.1367 CT, p.S456 L; c.1535 GA, p.R512 Q and c.1846_1847 del, p.Y616 Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-t RNA synthetase(h Arg RS), are linked to Pelizaeus-Merzbacher-like disease(PMLD) with unclear pathogenesis. Among these mutations, c.5 AG is the most extensively reported mutation, leading to a p.D2 G mutation in the N-terminal extension of the long-form h Arg RS. Here, we showed the detrimental effects of R512 Q substitution and ΔC mutations on the structure and function of h Arg RS, while the most frequent mutation c.5 AG, p.D2 G acted in a different manner without impairing h Arg RS activity. The nucleotide substitution c.5 AG reduced translation of h Arg RS m RNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.     

Expression,Solubilization and Purification of a Human Olfactory Receptor from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Trition X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.     

Mutation at the CK2 phosphorylation site on Cdc28 affects kinase activity and cell size in Saccharomyces cerevisiae

We have recently reported that protein kinase CK2 phosphortylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family. Received: 18 June 1997 / Accepted: 23 September 1997     

GOlf Complements A Gpa1 Null Mutation in Olf Saccharomyces Cerevisiae and Functionally Couples to the Ste2 Pheromone Receptor

Abstract

We have produced a plasmid designed for the expression of heterologous G protein α subunits in the yeast Saccharomyces cerevisiae Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein α subunit, to the yeast pheromone response pathway.

The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal- null mutation. A similar response is obtained when an alternative G protein a subunit, G_olf, is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.     

RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.     

The RAD52 gene is not required for the function of the DEL1 mutator gene in Saccharomyces cerevisiae

Summary The DEL1 mutator in Saccharomyces cerevisiae leads to the formation of deletions adjacent to itself (Liebman et al. 1979). Here we show that the frequency of these DEL1-promoted deletions is not altered by the presence of the recombination-deficient mutation, rad52-1. This indicates that generalized recombination is not required for the formation of deletions in DEL1 yeast strains.     

Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Abstract

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO^? detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Δyhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO^? generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Δyhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.     

Two novel ANK1 loss‐of‐function mutations in Chinese families with hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia disorder. ANK1 mutations account for most HS cases, but pathogenicity analysis and functional research have not been widely performed for these mutations. In this study, in order to confirm diagnosis, gene mutation was screened in two unrelated Chinese families with HS by a next‐generation sequencing (NGS) panel and then confirmed by Sanger sequencing. Two novel heterozygous mutations (c.C841T, p.R281X and c.T290G, p.L97R) of the ANK1 gene were identified in the two families respectively. Then, the pathogenicity of the two new mutations and two previously reported ANK1 mutations (c.C648G, p.Y216X and c.G424T, p.E142X) were studied by in vitro experiments. The four mutations increased the osmotic fragility of cells, reduced the stabilities of ANK1 proteins and prevented the protein from localizing to the plasma membrane and interacting with SPTB and SLC4A1. We classified these four mutations into disease‐causing mutations for HS. Thus, conducting the same mutation test and providing genetic counselling for the two families were meaningful and significant. Moreover, the identification of two novel mutations enriches the ANK1 mutation database, especially in China.     

Secretory and GM1 receptor binding role of N-terminal region of LTB in Vibrio cholerae

Heat labile enterotoxin from enterotoxigenic Escherichia coli is similar to cholera toxin (CT) and is a leading cause of diarrhea in developing countries. It consists of an enzymatically active A subunit (LTA) and a carrier pentameric B subunit (LTB). In the current study, we evaluated the importance of the N-terminal region of LTB by mutation analysis. Deletion of the glutamine (ΔQ3) residue and a substitution mutation E7G in the α1 helix region led to defects in LTB protein secretion. Deletion of the proline residue (ΔP2) caused a decrease in α helicity. The ΔP2 mutant affected GM₁ ganglioside receptor binding activity without affecting LTB pentamer formation. Upon refolding/reassembly, the ΔP2 mutant showed defective biological activity. The single substitution mutation (E7D) strengthened the helix, imparting structural stability and thereby improved the GM₁ ganglioside receptor binding activity. Our results demonstrate the important role of N-terminal α1 helix in maintaining the structural stability and the integrity of GM₁ ganglioside receptor binding activity.     

Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(?5/?5), Δ(?5/?4), Δ(?5/?3), Δ(?5/?2), and Δ(?5/?1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(?5/?1) denotes the deletion of residues numbered from ?5 to ?1 (TEFKA), while Δ(?5/?2) denotes the deletion of resides numbered from ?5 to ?2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(?5/?4) > WT > Δ(?5/?3) > Δ(?5/?5) > Δ(?5/?1) ~ Δ(?5/?2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]₂₂₂, a probe for detecting change in secondary structure, and Δε₄₀₅, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.     

The albinism of the feral Asinara white donkeys (Equus asinus) is determined by a missense mutation in a highly conserved position of the tyrosinase (TYR) gene deduced protein

A feral donkey population (Equus asinus), living in the Asinara National Park (an island north‐west of Sardinia, Italy), includes a unique white albino donkey subpopulation or colour morph that is a major attraction of this park. Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In this study, we analysed the donkey TYR gene as a strong candidate to identify the causative mutation of the albinism of these donkeys. The TYR gene was sequenced from 13 donkeys (seven Asinara white albino and six coloured animals). Seven single nucleotide polymorphisms were identified. A missense mutation (c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across kingdoms), which disrupts the first copper‐binding site (CuA) of functional protein, was identified in the homozygous condition (G/G or D/D) in all Asinara white albino donkeys and in the albino son of a trio (the grey parents had genotype C/G or H/D), supporting the recessive mode of inheritance of this mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had genotype G/G whereas all other coloured donkeys had genotype C/C or C/G. Across‐population association between the c.604C>G genotypes and the albino coat colour was highly significant (P = 6.17E?18). The identification of the causative mutation of the albinism in the Asinara white donkeys might open new perspectives to study the dynamics of this putative deleterious allele in a feral population and to manage this interesting animal genetic resource.     

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis‐sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography‐mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.