Expression profiling the human septin gene family

The septins are an evolutionarily conserved family of GTP-binding proteins involved in diverse processes including vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration, and neoplasia. The present paper reports a comprehensive study of septin gene expression by DNA microarray methods in 10 360 samples of normal, diseased, and tumour tissues. A novel septin, SEPT13, has been identified and is shown to be related to SEPT7. It is shown that SEPT13 and the other known human septins are expressed in all tissue types but some show high expression in lymphoid (SEPT1, 6, 9, and 12) or brain tissues (SEPT2, 3, 4, 5, 7, 8, and 11). For a given septin, some isoforms are highly expressed in the brain and others are not. For example, SEPT8_v2 and v1, 1* and 3 are highly expressed in the brain and cluster with SEPT2, 3, 4, 5, 7, and 11. However, a probe set specific for SEPT8_v1 with low brain expression clusters away from this set. Similarly, SEPT4 has lymphoid and non-lymphoid forms; SEPT2 has lymphoid and central nervous system (CNS) forms; and SEPT6 and SEPT9 are elevated in lymphoid tissues but both have forms that cluster away from the lymphoid forms. Perturbation of septin expression was widespread in disease and tumours of the various tissues examined, particularly for conditions of the CNS, where alterations in all 13 septin genes were identified. This analysis provides a comprehensive catalogue of the septin family in health and disease. It is a key step in understanding the role of septins in physiological and pathological states and provides insight into the complexity of septin biology.     

SEPT9 sequence alternations causing hereditary neuralgic amyotrophy are associated with altered interactions with SEPT4/SEPT11 and resistance to Rho/Rhotekin-signaling

SEPT9 is a member of the cytoskeleton-related septin family, which is highly expressed in glia cells in neuronal tissues. Sequence alterations in SEPT9 are known to cause hereditary neuralgic amyotrophy (HNA) but precise cellular consequences have yet to be determined. Since SEPT9 is thought to function through interaction with other septins and small GTPase Rho-mediated signaling, we analyzed the properties of HNA-associated SEPT9 missense variants, SEPT9F (c.278C>T/p.Ser93Phe in SEPT9_v3; NM_006640.3) and SEPT9W (c.262C>T/p.Arg88Trp in SEPT9_v3). We found both sequence variants, but not the wild type, to form filaments with SEPT4 along stress fibers in mesenchymal mouse mammary gland NMuMG cells. In the epithelial cells, the variants, but not the wild type, were colocalized with SEPT11 at cell-cell junctions. In addition, although septin filaments containing SEPT9_v3 were disrupted by Rho/Rhotekin signaling, this was not the case with SEPT9F and SEPT9W. Sequence variations in SEPT9 causing HNA are thus likely to alter modes of interaction with partner molecules in cells, and consequently contribute to the pathogenesis of HNA.     

SEPT9_i1 and genomic instability: mechanistic insights and relevance to tumorigenesis

Septins are highly conserved cytoskeletal GTP-binding proteins implicated in numerous cellular processes from apoptosis to vesicle trafficking. Septins have been associated with leukemia and solid tumor malignancies, including breast, ovarian, and prostate. We previously reported that high SEPT9_i1 expression in human mammary epithelial cell lines (HMECs) led to malignant cellular phenotypes such as increased cell proliferation, invasiveness, motility, and genomic instability. Our goal here was to better understand how SEPT9_i1 expression might contribute to genomic instability and malignant progression. First, we confirmed that even transient expression of SEPT9_i1 was sufficient to increase aneuploidy in HMECs. We then analyzed SEPT9_i1 by immunoprecipitation and immunofluorescence studies and found that SEPT9_i1 interacts with both α and γ tubulin. SEPT9_i1 expressing cells demonstrated dramatic chromosome segregation defects, centrosome amplification and cytokinesis defects, suggesting two possible molecular mechanisms contributing to the development of genomic instability. This suggests that SEPT9_i1 may promote genomic instability through both cytokinesis and mitotic spindle defects which lead to chromosome missegregation.     

Conquering the complex world of human septins: implications for health and disease

Peterson EA and Petty EM. Conquering the complex world of human septins: implications for health and disease. Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in must eukaryotes. Septins can bind and hydrolyze GTP, which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1‐SEPT14, which encode dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N‐terminus and/or C‐terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alterations of these proteins, by mutation or expression changes, have been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from alterations of expression in solid tumors or translocations in leukemias [mixed lineage leukemia (MLL)]. Expression changes in septins have also been associated with neurological conditions such as Alzheimer's and Parkinson's disease, as well as retinopathies, hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder hereditary neuralgic amyotrophy (HNA). Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype

The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10(-4)), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was examined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.     

Biochemical and genetic characterization of respiration-deficient mutants of Chinese hamster cells with a Gal− phenotype

Four Chinese hamster somatic cell mutants A13G9, 34A13G32, 2A13G14, and V6IG15 with a Gal^– phenotype have the following characteristics: (1) a low respiration rate; (2) a reduced Krebs cycle activity; (3) a low level of stimulation of oxygen consumption of mutant mitochondria by malate; (4) an absolute dependence on an ample supply of glucose to sustain a high rate of glycolysis; (5) a defect in the electron transport chain from NADH to coenzyme Q; and (6) no appreciable activity of rotenone-sensitive NADH oxidase in mutant mitochondria. These four mutants and another mutant, P12GX1, were analyzed by complementation analysis using seven other respiratory mutants of Dr. Scheffler which define seven complementation groups (I–VII). P12GX1 fails to complement mutant CCL16-B9 (group IV). A13G9 and 34A13G32 do not complement each other. Mutants V6IG15, A13G9, and 34A13G32 define two new groups of complementation (VIII and IX), while 2A13G14 does not complement mutants of groups II and VI.     

Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine metabolism. We identified and characterized 6 mutations, DelE85, K124R, A127V, Q145E, G152A, and E345V in 5 Spanish T2-deficient patients. Transient expression of mutant cDNAs was done at 37 and at 30 degrees C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37 degrees C and it retained 15% residual T2, indicating that specific activity of Q145E mutant protein was almost normal. This mutation reduced the heat stability of T2 activity. Although no significant residual activity was detected in either the G152A and A127V substitution, mutant proteins were detected, at 12.5% the normal amount at 37 degrees C and one-half normal at 30 degrees C for A127V, and 25 % only at 30 degrees C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30 degrees C expression were stable for 48 h at 37 degrees C after cycloheximide treatment. Expression of DelE85, K124R, and E345V cDNAs gave neither residual T2 protein nor T2 activity. We constructed an improved tertiary structural model of T2 based on the X-ray crystal structure of acetoacetyl-CoA thiolase of Zoogloea ramigera. On the basis of this model, K124, A127, and G152 are located near the active site, mutations of which might affect catalytic function whereas Q145E, De185E, and E345V are distant from the active site with mutants being expected to destabilize the tertiary structure, especially during protein folding and dimerization.     

Disease associations and altered immune function in CD45 138G variant carriers

The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance.     

SEPT12 mutations cause male infertility with defective sperm annulus

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.     

人隔蛋白7的真核表达载体的构建及表达

目的构建人隔蛋白7(SEPT7,hCDC10/SEPT7)的表达载体并对U251人脑恶性胶质瘤细胞系进行转染且检测其表达。方法RT-PCR方法扩增SEPT7cDNA片段,并将扩增的片段插入pCDNA3真核表达载体,构建成含SEPT7cDNA的重组载体pCDNA3/SEPT7,以脂质体介导该质粒转染人脑恶性胶质瘤细胞系U251,应用RT-PCR和免疫荧光染色鉴定转染细胞中hCDC10/SEPT7的表达。用流式细胞术对转染前后的U251细胞进行细胞周期分析。结果成功构建含SEPT7cDNA的重组载体pCDNA3/SEPT7,并使其稳定转染U251细胞,在转染的细胞中,证实有SEPT7mRNA表达上调。细胞周期检测结果G0/G1期细胞增多,SPF降低。结论成功构建SEPT7表达载体,并在人脑恶性胶质瘤细胞系获得表达,延迟细胞周期进展。     

SEPT7基因抑制人胶质瘤细胞系U251MG侵袭的研究

目的 研究SEPT7基因对人胶质瘤细胞系U251MG侵袭的抑制作用及其可能的分子机制.方法 以腺病毒为载体转导SEPT7(rAd5-SEFF7)入U251人脑胶质瘤细胞系;Transwell法和3-D Matrigel法观察U251胶质瘤细胞侵袭能力的变化,划痕实验和2-D Matrigel法观察细胞迁移能力的变化.应用蛋白印记检测MMP2,MMP9,MT1-MMP,TIMP1和TIMP2的表达变化,蛋白印记和免疫荧光检测整合素αvβ3的表达,以及应用激光扫描共聚焦显微镜观察细胞骨架蛋白tubulin-α结构的变化.结果 转染SEPT7后U251MG细胞的侵袭和迁移能力明显受到抑制、细胞MMP2、MMP9、MT1-MMP和整合素αvβ3的表达下调、TIMP1和TIMP2的表达则上调;肿瘤细胞的微管蛋白tubulin-α结构出现了重新分布,发生了扭曲及聚集现象,接近于正常的非肿瘤细胞的tubulin-α结构.结论 SEPT7基因可以抑制胶质瘤细胞的侵袭和迁移能力,其分子机制可能通过逆转MMPs/TIMPs的失衡状态,降低整合素αvβ3的表达,以及改变细胞骨架tubulin-α的结构而实现的.SEPT7可作为基因治疗胶质瘤的重要候选基因.     

Heterologous expression of wild-type and mutant β-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) β-cardiac myosin. After differentiation for 5–7 days, cWT or mutant β-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation ( t _0.5), the maximum velocity of shortening ( V _max) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT β-cardiac myosin significantly increased ttp and t _0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant β-cardiac myosin with those expressing cWT β-cardiac myosin, we found that ttp and t _0.5 were significantly decreased, and V _max was increased for the D778G mutant, whereas ttp, t _0.5 and V _max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.     

Cells expressing partially unfolded R789C/p.R989C type II procollagen mutant associated with spondyloepiphyseal dysplasia undergo apoptosis

We investigated the effects of the presence of R75C (p.R275C), R519C (p.719C), R789C (p.R989C), and G853E (p.G1053E) type II collagen (COL2A1) mutants, associated with distinct forms of spondyloepiphyseal dysplasia (SED), on the biological processes occurring in chondrocytic cells harboring those mutants. Mutant-specific biological responses of cells were initiated by activating tetracycline (Tet)-dependent expression of type II collagen mutants. Employing microscopic and biochemical assays, we determined that cells expressing the thermolabile R789C (p.R989C) type II collagen mutant undergo apoptosis. In contrast, in cells expressing the thermostable R75C (p.R275C), R519C (p.719C), and G853E (p.G1053E) mutants, apoptotic markers were not apparent. We also demonstrated that the R789C (p.R989C) mutant formed atypical complexes with endoplasmic reticulum (ER)-resident chaperones, thereby indicating an "unfolded protein response" (UPR) of cells harboring this specific mutant. Apoptotic changes were also demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and cleaved caspase 3 assays in the growth plates of mice harboring the R992C (p.R1147C) substitution in type II collagen. Based on these results, we propose that the intracellular presence of structurally altered type II collagen mutants could activate an apoptotic response, thereby limiting cell survival. By analyzing the response of cells to the altered structure of collagen mutants, our study contributes to better understanding the molecular basis of the pathological changes seen in vivo at the tissue level.     

Characterization of an avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phospholipases.

An avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis subsp. gelechiae, isolated by Heierson et al. (A. Heierson, I. Sidén, A. Kivaisi, and H. G. Boman, J. Bacteriol. 167:18-24, 1986) as a spontaneous phage-resistant mutant, was further characterized and found to lack the expression of phosphatidylcholine- and phosphatidylinositol-degrading phospholipase C, beta-lactamase, and flagellin because of the absence of corresponding mRNAs. The avirulent mutant was also found to be less efficient in killing insect cells in vitro than the wild type and to have altered behavior in vivo; wild-type B. thuringiensis does not circulate in the insect hemolymph after injection, whereas the avirulent mutant and nonpathogenic control bacteria remain in circulation. Flagella and motility may be important for virulence in the early stages of an infection; mutants with decreased motility appear less virulent when fed to Trichoplusia ni but not when injected. The 50% lethal doses of wild-type strain Bt13 and avirulent mutant strain Bt1302 were estimated to be 0.52 +/- 0.25 and 2,600 +/- 1,300 CFU per injected larva, respectively.     

Requirements of the membrane proximal tyrosine and dileucine-based sorting signals for efficient transport of the subtype C Vpu protein to the plasma membrane and in virus release

Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T cell loss following inoculation in macaques (Hill et al., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the membrane proximal tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of subtype C Vpu proteins, virus replication, and release.