Characteristics of IFITM,the newly identified IFN-inducible anti-HIV-1 family proteins

IFN-inducible IFITM proteins (IFITM1, 2, and 3) inhibit the replication of various viruses including HIV-1 through poorly understood mechanisms. Here, we further analyzed characteristics of these newly identified HIV-1 restriction factors. Firstly, in contrast to other anti-HIV-1 proteins, such as tetherin and APOBEC3G, IFITMs were resistant to a down-regulation of surface expression or degradation by HIV-1 proteins. Secondly, the enforced expression of IFITMs reduced the production of HIV-1 viruses from cells transfected with proviral plasmids containing whole viral sequences. Although their inhibitory activities were modest when compared to that of tetherin, IFITMs, but not tetherin, directly reduced the expression of HIV-1 proteins including Gag, Vif and Nef. Of importance, however, IFITMs had no inhibitory effect when these viral proteins were expressed by codon-optimized cDNAs that bypassed the viral-specific expression machinery. Indeed, our results supported the idea that IFITMs interfere with viral protein expression mediated by double-stranded viral RNAs, such as RRE and TAR. Finally, the S-palmitoylation of IFITMs, which is crucial for their anti-influenza virus activity, was not required for their anti-HIV-1 activity, indicating that IFITMs restrict these viruses at different steps. These characteristics lead to a better understanding of the mechanism by which IFITMs restrict HIV-1 and other viruses.     

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.     

Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185

GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.     

Identification and gene organization of three novel members of the IL-1 family on human chromosome 2

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.     

Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart

RTP/Drg1/Cap43/rit42/TDD5/Ndr1/NDRG1 (referred to as NDRG1 hereafter) is a cytoplasmic protein involved in stress responses, hormone responses, cell growth, and differentiation. Recently, the mutation of this gene was reported to be causative for hereditary motor and sensory neuropathy-Lom. Here, we cloned two human cDNAs encoding NDRG3 and NDRG4, which are homologous to NDRG1. These two genes, together with NDRG1 and a previously deposited cDNA (designated NDRG2), constitute the NDRG gene family. The four members share 57-65% amino acid identity. NDRG4 was further characterized because its mRNA expression was quite specific in brain and heart, in contrast to the relatively ubiquitous expression of the other three members. NDRG4 mRNA consists of three isoforms, NDRG4-B, NDRG4-B(var), and NDRG4-H. Northern and Western blot analyses showed that NDRG4-B was expressed only in the brain, whereas NDRG4-H was expressed in both brain and heart. NDRG4-B(var) was a minor product. NDRG4 expression was more abundant in adult than fetal brain and heart and was markedly decreased in the Alzheimer's diseased brain. In situ hybridization showed that NDRG4 was localized in neurons of the brain and spinal cord. The NDRG4 gene contains 17 exons. mRNA expression of the three NDRG4 isoforms is regulated by alternative splicing and possibly by alternative promoter usage. The finely tuned expression of the NDRG gene family members suggests that they have different specific functions.     

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.     

Differential interactions between Beclin 1 and Bcl-2 family members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x(L), Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x(L) was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x(L). Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x(L). Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x(L) and Beclin 1.     

Cloning and comparative sequence analysis of PUM1 and PUM2 genes,human members of the Pumilio family of RNA-binding proteins

Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.     

Bcl-2 family members and disease

Apoptosis plays an important role during development and in the maintenance of multicellular organisms. Bcl-2 family members affect cell death in either a positive or negative fashion. Although some redundancy exists between family members, expression of certain family members is important during development in an organ-specific manner. The founding family member bcl-2 tends to be highly expressed in the embryo and declines postnatally following differentiation and maturation. Altered expression of bcl-2, as well as other family members, has been observed in disease states potentially affecting treatment modalities. Here we examine the distribution and role death repressors bcl-2, bcl-x(L) and bcl-w as well as death effectors bax and bak play regulating apoptosis in a tissue-specific manner. Understanding the normal role of these proteins during embryogenesis and in the mature organ will give us important insight into what goes awry in various disease states.     

Regulation of human erythropoiesis by activin A,BMP2, and BMP4, members of the TGFbeta family

Activin A, BMP2, and BMP4, members of the TGFbeta family, have been implicated in the regulation of hematopoiesis. Here we explore and compare, for the first time in human primary cells, the role of activin A, BMP2, and BMP4 during erythropoiesis. Using in vitro erythroid differentiation of CD34(+) primary cells, we obtained the main stages of early erythropoiesis, characterized at the molecular, biochemical, and functional levels. Our results indicate that BMP2 acts on early erythroid cells and activin A on a more differentiated population. We report an insight into the mechanism of commitment of erythropoiesis by activin A and BMP2 involving two key events, increase in EPO-R and decrease in GATA2 expression. Simultaneous addition of activin A with BMP molecules suggests that BMP2 and BMP4 differently affect activin A induction of erythropoiesis. Follistatin and FLRG proteins downmodulate the effects of activin A and BMP2 on erythroid maturation.     

Genomic organization and chromosome localization of the newly identified human heparanase gene

Heparanase (HPSE), which we have recently isolated, is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this report, the genomic organization and chromosome localization of the human heparanase gene is described. Polymerase chain reaction, subcloning and DNA sequencing analysis of a bacterial artificial chromosome (BAC) clone revealed that the 3.7 kb human heparanase cDNA is spread over about 50 kb and contains 14 exons and 13 introns. The heparanase gene is expressed as two mRNA species containing the same open reading frame, HPSE 1a (5 kb) (GenBank Data Library under accession number: AF155510); and HPSE 1b (1.7 kb) (GenBank Data Library under accession number: AF144325), generated by alternative splicing. The HPSE 1a-form contains all 14 exons, whereas in the HPSE 1b-form the first and fourteenth exons (5'- and 3'-untranslated region) have been spliced out. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 13 (which is GA instead of GT), and the splice acceptor of intron 13 (which is GG instead of AG). Fluorescence in situ hybridization and radiation hybrid mapping suggest that the heparanase gene is located on human chromosome 4q22. This report regarding the structure of the human heparanase gene will aid in understanding the genetic contribution of this gene to normal physiology as well as to disease states. A possible involvement of heparanase in neuronal degeneration is discussed.     

Molecular characterization of the thermostability and carbohydrate-binding module from a newly identified GH118 family agarase,AgaXa

AgaXa is a thermostable β-agarase from the agar-degrading bacterium Catenovulum sp. X3. To further understand the mechanism of protein stabilization of AgaXa, several mutants were generated by random and site-directed mutagenesis, and they were subsequently screened by analysing their enzymatic activity and thermostability. Four mutants (V197D, P259H, C442S and C528S) were found for which the enzyme activity and thermostability were significantly decreased. Moreover, secondary structures determined by circular dichroism (CD) showed that mutants V197D and P259H presented a higher percentage of α-helix but fewer β-strands than wild-type (WT) AgaXa. On the contrary, no significant changes were observed in the secondary structures of mutants C442S and C528S. Through the treatment by proteinase K, different digested profiles were generated from mutants V197D and P259H when compared to WT, and mutants C442S and C528S was observed with more digested protein fragments. These results indicate that the enzymatic activity and stability of AgaXa is mainly related to its hydrophobic structure and disulphide bonds. Furthermore, carbohydrate-binding ability was also analysed for the mutants of N- and C-terminal deletions, and the results showed that agarose binding capability was not changed, indicating that the carbohydrate-binding module is probably located in the middle of the β-agarase AgaXa.     

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.     

The newly identified human nuclear protein NXP-2 possesses three distinct domains, the nuclear matrix-binding, RNA-binding, and coiled-coil domains

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.     

Bcl-2 family members and apoptosis, taken to heart

Loss of myocardial cells via apoptosis has been observed in many cardiovascular diseases and has been shown to contribute to the initiation and progression of heart failure. The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. These proteins decide whether the mitochondria should initiate the cell death program and release proapoptotic factors such as cytochrome c. The Bcl-2 proteins consist of anti- and proapoptotic members and play a key role in regulating apoptosis in the myocardium. The antiapoptotic proteins have been demonstrated to protect against various cardiac pathologies, whereas the antiapoptotic proteins have been reported to contribute to heart disease. This review summarizes the current understanding of the role of Bcl-2 proteins in the heart. cardiovascular disease; cytochrome c; protein; mitochondria