The Rad50 hook domain regulates DNA damage signaling and tumorigenesis

The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn²⁺-dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hook-dependent Mre11 complex functions. One of these alleles, Rad50⁴⁶, conferred reduced Zn²⁺ affinity and dimerization efficiency. Homozygous Rad50^46/46 mutations were lethal in mice. However, in the presence of wild-type Rad50, Rad50⁴⁶ exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50^+/46 exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50^+/46 Atm^+/− mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.     

Nuclear localization of Rad51B is independent of Rad51C and BRCA2

Rad51B is one of the five paralogs of human Rad51 and is found in a multiprotein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Participation of Rad51B in this complex depends on its direct interaction with Rad51C. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines reveal the nuclear localization of Rad51B. Mutations in the N-terminal KKLK motif of Rad51B (amino acids 4-7), result in the cytoplasmic localization of Rad51B suggesting that the KKLK sequence is the nuclear localization signal (NLS) for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in hamster irs3 mutant cells, deficient in Rad51C, showed that Rad51B localizes to the nucleus independently of Rad51C, the only known direct binding partner for Rad51B. Utilization of a BRCA2 mutant cell line, CAPAN-1, showed that Rad51B also localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate. This study finds that a KKLK motif in the N-terminus of Rad51B serves as an NLS that allows Rad51B to localize to the nucleus independent of Rad51C or BRCA2.     

A conserved leucine in the cytoplasmic domain of the Semliki Forest virus spike protein is important for budding

Summary.  Budding of alphaviruses at the plasma membrane has been shown to depend on specific amino acids of the spike protein and hydrophobic cavities of the nucleocapsid. Here the function of leucine401 in the cytoplasmic tail of the Semliki Forest virus spike protein was studied. When alanine and threonine were substituted for leucine the budding efficiency decreased. When the alanine mutant virus was passaged and sequenced a valine residue at position 401 was found which could partially restore budding proficiency. These results show that leucine401 together with the previously identified tyrosine399 form a motif that is required for budding. Received December 22, 1999 Accepted January 31, 2000     

新城疫病毒HN糖蛋白促细胞融合区域内保守氨基酸基因突变分析

目的 确定新城疫病毒(NDV)血凝素神经氨酸酶(HN)糖蛋白促细胞融合区域内的保守氨基酸功能,探讨HN糖蛋白的促细胞融合机制.方法 采用PCR定点突变与体内同源重组相结合的方法将HN糖蛋白促细胞融合区域内6个保守氨基酸突变为丙氨酸(A),在BHK21细胞内表达后,流式细胞仪定量分析蛋白的表达效率,并分别检测其促细胞融合活性、血细胞吸附能力(也称为受体识别活性)和神经氨酸酶活性.结果 L74A蛋白表达效率降低为72.7％,各突变株蛋白表达效率与野毒株相比差别无统计学意义(P＜0.05).各突变株蛋白促细胞融合活性都有不同程度的下降,其中I103A下降幅度最大,为野毒株的9.1％,各突变株蛋白血细胞吸附能力也都出现不同程度的降低,其中I1iA的下降最明显,为28.2％,各突变株蛋白的神经氨酸酶活性变化程度不同,其中174A略有升高,为118.6％,L110A下降幅度最大,为5.2％,I103A仅次于L1 10A,为5.7％.结论 新城疫病毒HN糖蛋白促细胞融合区域内的保守氨基酸在细胞融合中发挥着重要作用,第103位异亮氨酸(Ⅰ)是此区域内的关键氨基酸.     

Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation

Recovery from fast inactivation in voltage-dependent Na⁺ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICM_MTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na⁺ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes ∼20% to total gating charge ( Q _max), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICM_MTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICM_MTSET were recorded before and after modification with MTSEA_o. Prior to MTSEA_o, the time course of the gating charge during repolarization ( off -charge) was best described by a single fast time constant. After MTSEA, the off -charge had both fast and slow components, with the slow component accounting for nearly 35% of Q _max. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.     

Rad54 is dispensable for the ALT pathway

Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway.     

The Moraxella catarrhalis autotransporter McaP is a conserved surface protein that mediates adherence to human epithelial cells through its N-terminal passenger domain

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.     

C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes

VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR(6) of VlsE was present in all of these dog and human serum samples.     

The putative movement domain encoded by nepovirus RNA-2 is conserved in all sequenced nepoviruses

Summary Significant similarity among putative movement domains in polyproteins encoded by RNA-2 of six nepoviruses is described.     

C-reactive protein is associated with sleep disordered breathing independent of adiposity

STUDY OBJECTIVES: It is well established that medical conditions such as obesity and cardiovascular disease are associated with increased levels of inflammatory biomarkers such as C-reactive protein (CRP). Prior studies have produced inconsistent results regarding the association between sleep disordered breathing (SDB) and CRP, possibly due to the confounding effects of obesity or medical comorbidity. The present study examined the association between degree of SDB and level of CRP independent of prevalent medical conditions and obesity. DESIGN: Cross-sectional study. SUBJECTS AND SETTING: University-based clinical sample referred for diagnostic polysomnography. MEASUREMENTS AND RESULTS: The study sample consisted of 69 men (mean age 40 years; mean BMI of 31.2 kg/m2) free of prevalent medical conditions including hypertension, diabetes mellitus, and cardiovascular disease. Measurements of morning and evening CRP levels were performed along with full-montage polysomnography. Confounding due to obesity was assessed by adjustments for body mass index, waist circumference, and percent body fat. A strong association was found between degree of SDB and serum levels of CRP, with or without adjustment for age and several measures of adiposity. Between the lowest and highest quartiles of apnea-hypopnea index (AHI) the mean difference in adjusted level of CRP was 3.88 microg/ml (P < 0.001). Moreover, an independent association between serum CRP levels and nocturnal hypoxia was also observed, whereas no association was noted with parameters of sleep architecture. CONCLUSIONS: While more research is needed to elucidate causal pathways involving the effects of sleep-related hypoxia on low-grade systemic inflammation, the results of this study suggest that mechanisms other than adiposity per se could contribute to the inflammatory state seen in adults with SDB.     

The dendritic cell-derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum

Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.     

CRB1 has a cytoplasmic domain that is functionally conserved between human and Drosophila.

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs was that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. Here we describe an alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologues of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies.     

Characterization of Spodoptera litura multicapsid nucleopolyhedrovirus 38.7 k protein, which contains a conserved BRO domain

Homology analysis revealed that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) 38.7 k protein has 22-83% amino acid identities with Ecotropis obliqua NPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, S. exigua MNPV and S. littoralis MNPV 38.7 k proteins. Analysis of the relationship of these 38.7 k proteins indicated that they contain a conserved BRO-N domain, and SpltMNPV and SpliMNPV 38.7 k proteins also contain a motif found in all known viral and prokaryotic single-strand DNA binding proteins. RT-PCR results showed that SpltMNPV 38.7 k gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter motif (ATAAG). Western blot analysis revealed that the 38.7 k was expressed in infected S. litura cells as a 41 kDa form and this protein distributed in the nucleus of infected cells. Using a histone extraction protocol, SpltMNPV 38.7 k could be detected in the histone H1 fraction. Micrococcal nuclease treatment released SpltMNPV 38.7 k protein from the chromatin fraction, suggesting that its involvement in nucleosome structures. Furthermore, column chromatography using DNA-cellulose showed that SpltMNPV 38.7 k protein interacted with nucleic acids. It was proposed that SpltMNPV 38.7 k might function as a DNA-binding protein.     

Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more “permissive” cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.     

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7_39-98 localized mostly to the nucleus. The GST-11E7 and GST-11cE7_39-98 were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.     

HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing

Antigen presentation to CD4⁺ T cells involves intracellular antigen processing and loading of peptides onto newly synthesized major histocompatibility complex (MHC)-class II molecules. Some antigens, such as the lipid-bound, native form of myelin basic protein (LB-MBP) can also be presented by recycling of cell surface MHC class II molecules. The data reported here demonstrate that a preparation of highly purified, delipidated MBP (HP-MBP) follows yet another presentation pathway. Similar to LB-MBP, presentation of HP-MBP to HLA-DR1-restricted T cells was independent of HLA-DM, of newly synthesized proteins, and of invariant chain expression. However, in contrast to LB-MBP, presentation of HP-MBP was also independent of internalization of surface HLA-DR molecules. The different requirements for the presentation of the two molecular forms of MBP were further confirmed by use of the protease inhibitor E64: presentation of LB-MBP but not of HP-MBP was inhibited after treatment of target cells with E64. Furthermore, intact HP-MPB bound to isolated HLA-DR molecules in vitro with an association rate that was considerably faster than that of short peptides. These results show that presentation of HP-MBP is independent of intracellular processing and suggest that it may be presented to T cells by direct binding to surface HLA-DR molecules.