Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.     

Regulation of p27Kip1 by miRNA 221/222 in Glioblastoma

Levels of p27Kip1, a key negative regulator of the cell cycle, are often decreased in cancer. In most cancers, levels of p27Kip1 mRNA are unchanged and increased proteolysis of the p27Kip1 protein is thought to be the primary mechanism for its down-regulation. Here we show that p27Kip1 protein levels are also down-regulated by microRNAs in cancer cells. We used RNA interference to reduce Dicer levels in human glioblastoma cell lines and found that this caused an increase in p27Kip1 levels and a decrease in cell proliferation. When the coding sequence for the 3'UTR of the p27Kip1 mRNA was inserted downstream of a luciferase reporter gene, Dicer depletion also enhanced expression of the reporter gene product. The microRNA target site software TargetScan predicts that the 3'UTR of p27Kip1 mRNA contains multiple sites for microRNAs. These include two sites for microRNA 221 and 222, which have been shown to be upregulated in glioblastoma relative to adjacent normal brain tissue. The genes for microRNA 221 and microRNA 222 occupy adjacent sites on the X chromosome; their expression appears to be coregulated and they also appear to have the same target specificity. Antagonism of either microRNA 221 or 222 in glioblastoma cells also caused an increase in p27Kip1 levels and enhanced expression of the luciferase reporter gene fused to the p27Kip1 3'UTR. These data show that p27Kip1 is a direct target for microRNAs 221 and 222, and suggest a role for these microRNAs in promoting the aggressive growth of human glioblastoma.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

Identification and characterization of DUSP27, a novel dual-specific protein phosphatase

A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4-14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50 kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism.     

Cloning, mapping and characterization of the human RAB27A gene

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.     

AtXTH27 plays an essential role in cell wall modification during the development of tracheary elements

Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.     

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the human cyclin-dependent kinase inhibitor p27kip1 in primary breast carcinomas

The human p27^kip1 gene encodes a cyclin-dependent kinase inhibitor implicated in the negative regulation of the cell cycle. In order to elucidate the possible role of p27 mutations in the development or progression of human breast cancer, we have studied the occurrence of genetic abnormalities in this gene in a series of 30 primary breast carcinomas. Direct sequence analysis of the polymerase chain reaction amplified human p27 gene revealed the occurrence of two sequence variations with respect to the reported sequence; both variants were also present in the lymphocyte DNA from the same patients. First, a silent G to A change at codon 142 (Thr) was detected in a single case. Second, a T to G transversion at codon 109, resulting in a Val to Gly change, was observed in eight tumour DNA samples (26%) and in 31 out of 80 unrelated normal individuals (39%). This latter change creates a BglI restriction site that might be useful for genetic analysis of human tumours. Despite the occurrence of these polymorphisms, we did not however find any evidence of somatic mutations in the coding region of the p27 gene. On the basis of these results, we suggest that alterations in the integrity of the human p27 gene are not common events in human breast carcinomas.     

Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.