A-kinase-interacting protein localizes protein kinase A in the nucleus

The genetic variability and covalent modifications associated with the amino terminus of the protein kinase A (PKA) catalytic (C) subunit suggest that it may contribute to protein-protein interactions and/or localization. By using a yeast two-hybrid screen, we identified a PKA-interacting protein (AKIP1) that binds to the amino terminus (residues 1-39) of the C subunit of PKA. The interaction was localized to the A helix (residues 14-39) of the C subunit and to the carboxyl terminus of AKIP1. AKIP1 thus defines the amino-terminal A helix of PKA as a protein interaction motif. In normal breast (Hs 578 Bst) and HeLa cells, AKIP1 is present in the nucleus as speckles. A nuclear localization signal (Arg-14 and Arg-15) was identified. Upon stimulation with forskolin, HeLa cells expressing AKIP1 accumulated higher levels of the endogenous C subunit in the nucleus. Deletion of the carboxyl terminus of AKIP1 or overexpression of residues 1-39 of the C subunit abolished nuclear localization of the activated endogenous C subunit. Thus, AKIP1 describes a PKA-interacting protein that can contribute to localization by a mechanism that is distinct from A-kinase anchoring proteins that interact with the regulatory subunits.     

DNA repair by the cryptic endonuclease activity of Mu transposase

Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common θ strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the θ intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835–3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or “5′flap” after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway.     

In vivo genetic mutations define predominant functions of the human T-cell leukemia/lymphoma virus p12I protein

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) ORF-I encodes a 99-amino acid hydrophobic membrane protein, p12(I), that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12(I). The removal of a noncanonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12(I) is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8-kDa protein. The 8-kDa protein in turn traffics to the cell surface, is recruited to the immunologic synapse following T-cell receptor (TCR) ligation, and down-regulates TCR proximal signaling. The uncleaved 12-kDa form of p12(I) resides in the ER and interacts with the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that affect proteolytic cleavage, suggesting that ER-associated functions of p12(I) may contribute to the survival and proliferation of the infected T cells in the host.     

丙型肝炎病毒核心蛋白DNA结合区的测定

目的 阐明丙型肝炎病毒（HCV）核心蛋白的DNA结合特性及其意义。方法 在大肠杆菌中表达谷胱甘肽转移酶（GST）融合HCV核心蛋白不同长度片段,并进行纯化。经聚丙烯酰胺凝胶电泳后,通过更换缓冲液的方法将这些蛋白片段相对固定在凝胶中,用γ－^32P[4667-0091]ATP标记人工合成的DNA进行2次电泳,通过放射自影检测核心蛋白的DNA结合区。结果 HCV核心蛋白N端第10－16位氨基酸残基和第46－70位氨基酸残基可独立地结合DNA,核心蛋白既能和单链DNA结合又能和双链DNA结合,对单双链DNA的结合域相同。对所结合的DNA序列无选择性。结论 HCV核心蛋白的N端含有两个DNA结合区,结合区序列与核内转移信号的部分重迭以及对结合靶DNA序列的非选择性可能是HCV核心蛋白具有多功能的基础。     

Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer. METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed. RESULTS: TP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P=0.0006). CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.     

Analysis of K-ras codon 12 mutations and p53 overexpression in colorectal nodule-aggregating tumors

BACKGROUND AND AIMS: Morphologically, colorectal nodule-aggregating tumors are quite different from polypoid-type colorectal tumors that develop via the adenoma-carcinoma sequence. Although polypoid-type colorectal tumors are well known to have a high incidence of K-ras gene mutation and p53 overexpression, colorectal nodule-aggregating tumors have not been examined in terms of genetic changes and clinicopathological features. In the present study, therefore, we analysed the clinicopathological features, genetic changes in K-ras codon 12, and p53 overexpression in colorectal nodule-aggregating tumors. METHODS: A total of 18 colorectal nodule-aggregating tumors were surgically resected and then analysed clinicopathologically. Immunohistochemistry and polymerase chain reaction-single stranded conformational polymorphism were performed to analyse p53 abnormalities in the tumors. K-ras codon 12 mutations were screened out by the polymerase chain reaction-restriction fragment length polymorphism method and analysed by fluorescence direct sequencing. RESULTS: p53 overexpression was observed in six lesions (33%). p53-overexpressing cells were observed in parts of carcinoma or adenoma showing high-grade atypia. Four of the 10 (40%) samples had a p53 gene mutation. Nine of the 18 (50%) samples had a K-ras codon 12 point mutation. In eight cases (89%), the mutations of the K-ras codon 12 were of the same type: GGT (glycine) to GTT (valine). CONCLUSIONS: The colorectal nodule-aggregating tumor has distinctive characteristics showing a morphological phenotype of the superficial-type tumors and genotype of the polypoid tumors in terms of K-ras gene mutation and p53 overexpression.     

Conformational effects of environmentally induced, cancer-related mutations in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. A considerable number of environmentally induced, cancer-related p53 mutations in human tumors have been found in a highly conserved proline-rich sequence of the p53 protein encompassed by amino acid residues 147-158. Using conformational energy analysis based on ECEPP (Empirical Conformational Energy for Peptides Program), we have determined the low-energy three-dimensional structures for this dodecapeptide sequence for the human wild-type p53 protein and three environmentally induced, cancer-related mutant p53 proteins with His-151, Ser-152, and Val-154, respectively. The results suggest that the wild-type sequence adopts a well-defined low-energy conformation and that the mutant peptides adopt well-defined conformations that are distinctly different from the conformation of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of these environmentally induced, cancer-related, mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4-8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki-67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53-positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell. In conclusion, strong IHC reactivity for p53 protein is not an indicator of the presence of p53 gene mutations at exons 4-8 in HCC. Thus, p53 loss of function in HCC should be evaluated both by p53 mutation analysis and p53 protein expression, as both give complementary information about p53 status.     

Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene mutations

Zebrafish carrying heterozygous mutations for 17 different ribosomal protein (rp) genes are prone to developing malignant peripheral nerve sheath tumors (MPNSTs), a tumor type that is seldom seen in laboratory strains of zebrafish. Interestingly, the same rare tumor type arises in zebrafish that are homozygous for a loss-of-function point mutation in the tumor suppressor gene p53. For these reasons, and because p53 is widely known to be mutated in the majority of human cancers, we investigated the status of p53 in the rp^+/− MPNSTs. Using monoclonal antibodies that we raised to zebrafish p53, we found that cells derived from rp^+/− MPNSTs are significantly impaired in their ability to produce p53 protein even in the presence of a proteasome inhibitor and γ-irradiation. Although the coding regions of the p53 gene remain wild type, the gene is transcribed, and overall protein production rates appear normal in rp^+/− MPNST cells, p53 protein does not get synthesized. This defect is observed in all MPNSTs we examined that were derived from our 17 zebrafish lines with rp gene mutations. To date, studies of p53 in malignancies have focused predominantly on either p53 gene mutations or the aberrant posttranslational regulation of the p53 protein. Our results show that the appropriate amount of numerous ribosomal proteins is required for p53 protein production in vivo and that disruption of this regulation most likely contributes to tumorigenesis.     

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1–73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.     

原发性小肠腺癌p16蛋白及PCNA的表达

目的检测p16蛋白和PCNA在原发性小肠腺癌组织、癌旁组织及正常小肠组织中的表达。方法采用SP免疫组化法对36例小肠腺癌及其相应癌旁组织、6例正常小肠组织进行p16蛋白及PCNA的定位观察。结果①原发性小肠腺癌组织中p16蛋白阳性率为88.89％，相应癌旁组织及正常小肠组织中p16蛋白未见表达；原发性小肠腺癌组织中p16蛋白的表达与其分化程度及有无淋巴结转移相关（P〈0.05）；②原发性小肠腺癌组织中PCNA阳性率为61.11％，相应癌旁组织及正常组织PCNA阳性率为28.57％，二者比较P〈0.05；原发性小肠腺癌组织中PCNA的表达与肿瘤部位、浸润深度、分化程度相关（P〈0.05）；③原发性小肠腺癌组织中p16蛋白的表达优于PCNA的表达（P〈0.05）。结论①小肠腺癌中p16蛋白的高表达可能是其癌变过程中的早期事件，有望作为小肠腺癌的免疫标志物；②PCNA可作为判断预后的有效指标；③原发性小肠腺癌组织中p16蛋白的表达优于PCNA的表达，两者联合检测可早期诊断小肠腺癌，判断预后。     

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance.     

Detection, rescue, and mapping of mutations in the adenovirus DNA binding protein gene

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in HindIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between EcoRI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and BamHI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202 EcoRI fragment A was crossed with the Ad5wt BamHI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.     

Aberrant p53 protein expression and function in a panel of hematopoietic cell lines with different p53 mutations

The p53 gene is one of the most important genes involved in carcinogenesis and its role in part has been clarified by research using cell lines. To know the comprehensive characteristics of 22 hematopoietic cell lines (T, 13 and non‐T, nine lines), the relationship between p53 mutational status, its altered functioning, and its mRNA and protein levels were examined. p53 mutations were less frequent in T‐cell lines (38% vs. 78%) with mainly single nucleotide substitutions generating missense codons. Of 22 different p53 mutations, 12 (54.5%) resulted in mutated proteins, with the mutations clustering mainly in the sequence‐specific DNA‐binding site region located from amino acid residues 102 to 292. p53 mRNA and protein assays determined that wild‐type cell lines expressed constant levels of both mRNA and protein, but mutated cell lines demonstrated two expression patterns: protein over‐expression with reduced mRNA levels, because of missense mutations; and protein under‐expression with little mRNA expression, because of other mutations. The resistance to Nutlin (MDM2 inhibitor)‐induced apoptosis was associated with p53 mutations independently of MDM2 expression levels. This clarification of the unique associations in cell lines useful for bio‐medical studies will contribute to a better understanding of p53‐associated carcinogenesis.     

Role of HSV-1 Capsid Vertex-Specific Component (CVSC) and Viral Terminal DNA in Capsid Docking at the Nuclear Pore

Penetration of the viral genome into a host cell nucleus is critical for initiation of viral replication for most DNA viruses and a few RNA viruses. For herpesviruses, viral DNA ejection into a nucleus occurs when the capsid docks at the nuclear pore complex (NPC) basket with the correct orientation of the unique capsid portal vertex. It has been shown that capsid vertex-specific component (CVSC) proteins, which are located at the twelve vertices of the human herpes simplex virus type 1 (HSV-1) capsid, interact with nucleoporins (Nups) of NPCs. However, it remained unclear whether CVSC proteins determine capsid-to-NPC binding. Furthermore, it has been speculated that terminal DNA adjacent to the portal complex of DNA-filled C-capsids forms a structural motif with the portal cap (which retains DNA in the capsid), which mediates capsid-NPC binding. We demonstrate that terminal viral DNA adjacent to the portal proteins does not present a structural element required for capsid-NPC binding. Our data also show that level of CVSC proteins on the HSV-1 capsid affects level of NPC binding. To elucidate the capsid-binding process, we use an isolated, reconstituted cell nucleus system that recapitulates capsid-nucleus binding in vivo without interference from trafficking kinetics of capsids moving toward the nucleus. This allows binding of non-infectious capsid maturation intermediates with varying levels of vertex-specific components. This experimental system provides a platform for investigating virus–host interaction at the nuclear membrane.     

K-ras codon 12 mutations of the super-minute dysplasia in Barrett''s esophagus by DNA extraction using a microdissection method

The aim of this study was to clarify the histogenesis of Barrett's cancer. First, 28 lesions of the super-minute dysplasia AGT), and 5 among 25 lesions with an average p53-LI of 58%, which were considered to be morphologically low grade dysplasia, showed K-ras mutation (four lesions: GGT-->GAT, 1 lesion: GGT-->AGT). This current study shows that some dysplasia lesions have K-ras mutations in their initial condition, whether these atypical tubule lesions are low grade dysplasia or high grade dysplasia (intramucosal adenocarcinoma), and supports the dysplasia-carcinoma sequence in the histogenesis of Barrett's cancer and synchronously suggests that there is a different route to it.     

肝细胞癌组织HSP70、p53和PCNA的表达及其意义

目的探讨热休克蛋白70（HSP70）、p53和增殖细胞核抗原（PCNA）在肝细胞癌（HCC）组织中的表达及其意义。方法采用免疫组化法检测正常人、慢性乙型肝炎、肝硬化和HCC肝或癌组织HSP70、p53和PCNA的表达情况。结果 HCC组织HSP70、p53和PCNA表达阳性率明显高于非癌组织（x1^2=27.16x2^2=67.6,x3^2=40.6,P〈0.01）;HSP70在正常人、慢性乙型肝炎、肝硬化和HCC中的表达逐步增强;HSP70和p53在分化较好的HCC中的阳性率明显低于分化不良者（x1^2=6.8,P1〈0.01x2^2=6.1,P2〈0.05）,而PCNA表达与HCC组织分化程度无关（x2=2.4,P〉0.05）;HSP70表达强度与p53和PCNA表达关系密切（x1^2=41.3,x2^2=41.4,P〈0.01）。结论 HCC是HSP70高表达肿瘤。HSP70表达与p53和PCNA表达密切相关,因而在HCC发生和发展中起重要作用。     

Immune responses in mice to DNA vaccination using the C-terminus of p43(p12) from Mycobacterium avium subspcies paratuberculosis

AIM To incorporate p12 in a plasmid under the control of the CMV promotor and test for the ability of theconstruct to produce specific immune responses in DNA-immunized mice.METHODS A His-tag fusion of the protein p12, was expressed in the prokaryotic expression vector (pQE)and the recombinant protein purified using nickel-chelate chromatography. His-tagged p12 was sub-clonedinto the pBK-CMV vector for expression in eukaryotic systems. Groups of six female balb/c mice werevaccinated with either 50μg im of the DNA pBK-CMV-p12 or pBK-CMV vector alone at week 0, andboosted at 2 and 4 weeks. ELISPOT assays (detection of p12 T-cell dependant IF-γ release) on mouse spleniccells were used to measure cell mediated immune responses and anti-mouse IgG ELISAs to detect antibodyresponse.RESULTS Significant CMI and humoral immune responses to recombinant p12 were detected in micevaccinated with pBK-CMV-p12 vector compared to mice vaccinated with pBK-CMV vector alone. The miceremained well throughout the development of immunity to p12.CONCLUSION A DNA vaccine coding for a specific MAP protein will stimulate humoral and cell mediatedimmune responses in mice.