Haplotype Analysis of the Human Endogenous Retrovirus Locus HERV-K(HML-2.HOM) and Its Evolutionary Implications

We and others recently identified an almost-intact human endogenous retrovirus (HERV), termed HERV-K(HML-2.HOM), that is usually organized as a tandem provirus. Studies on HERV proviral loci commonly rely on the analysis of single alleles being taken as representative for a locus. We investigated the frequency of HERV-K(HML-2.HOM) single and tandem alleles in various human populations. Our analysis revealed that another HERV-K(HML-2) locus, the so-called HERV-K(II) provirus, is also present as a tandem provirus allele in the human population. Proviral tandem formations were identified in various nonhuman primate species. We furthermore examined single nucleotide polymorphisms (SNPs) within the HERV-K(HML-2.HOM) proviral gag, prt, and pol genes, which all result in nonsense mutations. We identified four proviral haplotypes displaying different combinations of gag, prt, and pol SNPs. Haplotypes harboring completely intact proviral genes were not found. For the left provirus of the tandem arrangement a haplotype displaying intact gag and prt genes and a mutated pol was found in about two-thirds of individuals from different ethnogeographic origins. The same haplotype was always found in the right provirus. The various haplotypes point toward multiple recombination events between HERV-K(HML-2.HOM) proviruses. Based on these findings we derive a model for the evolution of the proviral locus since germ line integration. [Reviewing Editor : Dr. Martin Kreitman]     

Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses

Background

Integration of retroviral DNA into a germ cell may lead to a provirus that is transmitted vertically to that host's offspring as an endogenous retrovirus (ERV). In humans, ERVs (HERVs) comprise about 8% of the genome, the vast majority of which are truncated and/or highly mutated and no longer encode functional genes. The most recently active retroviruses that integrated into the human germ line are members of the Betaretrovirus-like HERV-K (HML-2) group, many of which contain intact open reading frames (ORFs) in some or all genes, sometimes encoding functional proteins that are expressed in various tissues. Interestingly, this expression is upregulated in many tumors ranging from breast and ovarian tissues to lymphomas and melanomas, as well as schizophrenia, rheumatoid arthritis, and other disorders.

Results

No study to date has characterized all HML-2 elements in the genome, an essential step towards determining a possible functional role of HML-2 expression in disease. We present here the most comprehensive and accurate catalog of all full-length and partial HML-2 proviruses, as well as solo LTR elements, within the published human genome to date. Furthermore, we provide evidence for preferential maintenance of proviruses and solo LTR elements on gene-rich chromosomes of the human genome and in proximity to gene regions.

Conclusions

Our analysis has found and corrected several errors in the annotation of HML-2 elements in the human genome, including mislabeling of a newly identified group called HML-11. HML-elements have been implicated in a wide array of diseases, and characterization of these elements will play a fundamental role to understand the relationship between endogenous retrovirus expression and disease.     

超级杂交稻乙烯反应元件结合蛋白cDNA的生物信息学分析与克隆

目的：从超级杂交稻中克隆乙烯反应元件结合蛋白（EREBP）的cDNA。方法：利用模式植物拟南芥中编码乙烯反应元件结合蛋白的cDNA,对现有的水稻基因组数据库进行搜索,获得一条高同源的未知序列。对这条未知序列的核酸序列蛋白质序列及其结构、性质、功能等进行生物信息学分析后,以超级杂交稻为材料,用未知序列设计一对简并引物,用RTPCR技术扩增后进行T-A克隆。结果：生物信息学分析结果表明,这个未知序列应为水稻中编码EREBP的cDNA;克隆后经测序获得一条915bp的cDNA,BLAST表明这条cDNA序列与未知序列的部分核酸序列的同源性达到了99％;提NCBI的GenBank后被接受,登录号为EF507537。结论：以生物信息学分析为基础,结合RT-PCR和T-A克隆技术,成功地从超级杂交稻中克隆了EREBP cDNA。     

A 30-kDa Protein in the Surface Complex and Flagella of Euglena has Protein Kinase Activity

ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-³²P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn²⁺ or Mg² and was inhibited or had greatly reduced activity with 5 mM Ca²⁺, Co^2-, Cu²⁺ or Zn²⁺. Incorporation was much lower when [γ-³²P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-³²P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [³²P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.     

Interface Analysis of the Complex between ERK2 and PTP-SL

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex.     

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane protein-associated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.     

Assembly and Kinetic Folding Pathways of a Tetrameric β-Sheet Complex: Molecular Dynamics Simulations on Simplified Off-Lattice Protein Models

We have performed discontinuous molecular dynamic simulations of the assembly and folding kinetics of a tetrameric β-sheet complex that contains four identical four-stranded antiparallel β-sheet peptides. The potential used in the simulation is a hybrid Go-type potential characterized by the bias gap parameter g, an artificial measure of a model protein's preference for its native state, and the intermolecular contact parameter η, which measures the ratio of intermolecular to intramolecular native attractions. The formation of the β-sheet complex and its equilibrium properties strongly depend on the size of the intermolecular contact parameter η. The ordered β-sheet complex in the folded state and nonaligned β-sheets or tangled chains in the misfolded state are distinguished by measuring the squared radius of gyration and the fraction of native contacts Q. The folding yield for the folded state is high at intermediate values of η, but is low at both small and large values of η. The folded state at small η is liquid-like, but is solid-like at both intermediate and large η. The misfolded state at small η contains nonaligned β-sheets and tangled chains with poor secondary structure at large η. Various folding pathways via dimeric and trimeric intermediates are observed, depending on η. Comparison with experimental results on protein aggregation indicates that intermediate η values are most appropriate for modeling fibril formation and small η values are most appropriate for modeling the formation of amorphous aggregates.     

A/U富集片段RNA结合蛋白的固相纯化与鉴定

RNA与细胞靶蛋白结合是RNA发挥其生物学功能的重要基础,因此,分离和鉴定RNA结合蛋白是研究RNA功能的必要步骤．目前,RNA结合蛋白的分离和鉴定方法较多,但各有优缺点．本实验利用可溶性碳二亚胺(EDC)介导的缩合反应,将A/U富集片段RNA共价偶联到固相介质amine M-270上,再用固定化RNA经亲和层析从细胞抽提物中分离纯化RNA结合蛋白,并以SDS-PAGE联合质谱分析和Western blotting等方法鉴定RNA特异性结合蛋白．最后通过荧光原位杂交和共聚焦显微镜证明这些RNA特异性结合蛋白确与RNA在细胞内结合．实践证明这一方法简单、高效、易于掌握．     

Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.     

Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca²⁺ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca²⁺ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca²⁺. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes.