Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH₂-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH₂-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271–285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796–1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Δ rce1Δ) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Δ single mutant, the ste24Δ single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH₂-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin–a-factor fusions to separate the NH₂- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Δ rce1Δ) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.     

Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10

Neprilysin (NEP) consists of 749 amino acids with two conserved cysteines (734, 746) and a putative CAAX motif (residues 746-749, CRVW) at the C-terminus. To investigate the role of the C-terminal conserved cysteine residues, three NEP mutants (C734S, C746S, and double mutant C734S/C746S) were constructed by use of site-directed mutagenesis. Western blot analysis of lysates of transfected cells revealed the presence of three NEP forms in wild type and mutants with a different glycosylation pattern. Point mutations of C734 as well as C746 by serine dramatically diminished the plasma membrane association of NEP as detected by flow cytometry and laser scanning microscopy. Endoprotease enzyme activity was slightly diminished in the C746S-NEP variant and was not detectable in the C734S-form of NEP suggesting a pivotal role of the C734 in the proper folding of the enzyme. Prenylation of NEP was not detected in an in vivo assay.     

Prenylation of viral proteins by enzymes of the host: Virus‐driven rationale for therapy with statins and FT/GGT1 inhibitors

Intracellular bacteria were recently shown to employ eukaryotic prenylation system for modifying activity and ensuring proper intracellular localization of their own proteins. Following the same logic, the proteins of viruses may also serve as prenylation substrates. Using extensively validated high‐confidence prenylation predictions by PrePS with a cut‐off for experimentally confirmed farnesylation of hepatitis delta virus antigen, we compiled in silico evidence for several new prenylation candidates, including IRL9 (CMV) and few other proteins encoded by Herpesviridae, Nef (HIV‐1), E1A (human adenovirus 1), NS5A (HCV), PB2 (influenza), HN (human parainfluenza virus 3), L83L (African swine fever), MC155R (molluscum contagiosum virus), other Poxviridae proteins, and some bacteriophages of human associated bacteria. If confirmed experimentally, these findings may aid in dissection of molecular functions of uncharacterized viral proteins and provide a novel rationale for statin and FT/GGT1‐based inhibition of viral infections. Prenylation of bacteriophage proteins may aid in moderation of microbial infections.     

Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens

The protein Bc059385, whose solution structure is reported here, is the human representative of a recently identified family of membrane-anchored ubiquitin-fold (MUB) proteins. Analysis of their similarity to ubiquitin indicates that homologous amino acid residues in MUBs form a hydrophobic surface very similar to the recognition patch surrounding Ile-44 in ubiquitin. This suggests that MUBs may interact with proteins containing an alpha-helical motif similar to those of some ubiquitin binding domains. A disordered loop common to MUBs may also provide a second protein interaction site. From the available data, it is probable that this protein is prenylated and associated with the membrane. With <20% identity to ubiquitin, the MUB family further expands the sequence space that maps to the beta-grasp fold, and adds membrane localization to its list of functional roles.     

Structure-based binding between protein farnesyl transferase and PRL-PTP of malaria parasite: an interaction study of prenylation process in Plasmodium

Protein prenylation is a post-translational modification critical for many cellular processes such as DNA replication, signaling, and trafficking. It is mediated by protein farnesyltransferase by recognizing ‘CAAX’ motif on protein substrate. Plasmodium falciparum also possesses many such proteins with ‘CAAX’ motif, involved in various pathways of the parasite. The interaction studies of PfPFT with its substrate were carried out using synthetic peptides but not with full protein. Therefore, in this study, we have modeled both PfPFT and its substrate protein tyrosine phosphatase (PfPRL-PTP) followed by interaction studies using protein–protein docking and molecular dynamics simulation. Our findings provided a clear picture of interactions at atomic level between prenyltransferase and its protein substrate. We are assured that this piece of information can be extended to many other proteins of parasite containing ‘CAAX’ motif and that it may also lead to the development of anti-malarials based on the inhibition of prenylation-dependent pathways of parasite..     

Molecular docking and structure-based virtual screening studies of potential drug target,CAAX prenyl proteases,of Leishmania donovani

Targeting CAAX prenyl proteases of Leishmania donovani can be a good approach towards developing a drug molecule against Leishmaniasis. We have modeled the structure of CAAX prenyl protease I and II of L. donovani, using homology modeling approach. The structures were further validated using Ramachandran plot and ProSA. Active site prediction has shown difference in the amino acid residues present at the active site of CAAX prenyl protease I and CAAX prenyl protease II. The electrostatic potential surface of the CAAX prenyl protease I and II has revealed that CAAX prenyl protease I has more electropositive and electronegative potentials as compared CAAX prenyl protease II suggesting significant difference in their activity. Molecular docking with known bisubstrate analog inhibitors of protein farnesyl transferase and peptidyl (acyloxy) methyl ketones reveals significant binding of these molecules with CAAX prenyl protease I, but comparatively less binding with CAAX prenyl protease II. New and potent inhibitors were also found using structure-based virtual screening. The best docked compounds obtained from virtual screening were subjected to induced fit docking to get best docked configurations. Prediction of drug-like characteristics has revealed that the best docked compounds are in line with Lipinski’s rule. Moreover, best docked protein–ligand complexes of CAAX prenyl protease I and II are found to be stable throughout 20 ns simulation. Overall, the study has identified potent drug molecules targeting CAAX prenyl protease I and II of L. donovani whose drug candidature can be verified further using biochemical and cellular studies.     

Molecular dissection of Drosophila Prickle isoforms distinguishes their essential and overlapping roles in planar cell polarity

Prickle-Spiny-Legs (Pk) is an essential component of the planar cell polarity (PCP) pathway, together with Frizzled (Fz) and Dishevelled (Dsh). A role for Pk was proposed to mediate feedback amplification of asymmetric Fz/Dsh activity across cell boundaries, ensuring a single prehair initiates at each distal vertex. Here we show that apical localisation of Pk^Pk and Pk^Sple isoforms are mutually independent and regulated by the C-terminal domain. The N-terminus of Pk^Pk is dispensable for PCP, whereas the unique N-terminal domain of Pk^Sple contains an additional localisation function, which confers a qualitatively different activity. Our results suggest that endogenous Pk^Pk and Pk^Sple can affect each other's function via the C-terminal domain, yet may not form heteromeric complexes. Overexpressing PET domain-deleted Pk variants interferes with a branch of Fz/Dsh signalling that regulates the number of wing hairs, and blocks non-cell-autonomous repolarisation. We infer that Pk^Pk is sufficient to mediate the intercellular feedback signalling. Significantly, Pk^Pk but not Pk^Sple is required for hexagonal cell packing in the pupal wing. We propose that Fz-dependent PCP readout reflects short-range, cell-contact based, interactions between hexagonal cells, rather than a direct response to an as yet unidentified diffusible ligand.     

Isolation of Pheromone Precursor Genes of Magnaporthe grisea

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.