Presenilin 1 protein directly interacts with Bcl-2.

Presenilin proteins are involved in familial Alzheimer's disease, a neurodegenerative disorder characterized by massive death of neurons. We describe a direct interaction between presenilin 1 (PS1) and Bcl-2, a key factor in the regulation of apoptosis, by yeast two-hybrid interaction system, by co-immunoprecipitation, and by cross-linking experiments. Our data show that PS1 and Bcl-2 assemble into a macromolecular complex, and that they are released from this complex in response to an apoptotic stimulus induced by staurosporine. The results support the idea of cross-talk between these two proteins during apoptosis.     

SARS-CoV accessory protein 7a directly interacts with human LFA-1

The SARS-CoV accessory protein 7a is a type I membrane protein with an extracellular domain of 81 amino acid residues. It is described to be expressed during infection and to be a component of the virus particle surface. In this study, we demonstrate that protein 7a binds directly and specifically to human lymphocyte function-associated antigen 1 (LFA-1) on the cell surface of Jurkat cells. The binding is increased upon artificial cell activation with phorbol ester. These observations are confirmed by direct in vitro binding of recombinant protein 7a to the wild type and mutant K287C/K294C I domain showing that the I domain is the 7a binding site in the alpha(L) chain of LFA-1. Consequences of the LFA-1 interaction with 7a are discussed. In particular, our data suggest LFA-1 to be an attachment factor or the receptor for SARS-CoV on human leukocytes.     

LTBP-2 specifically interacts with the amino-terminal region of fibrillin-1 and competes with LTBP-1 for binding to this microfibrillar protein.

LTBP-2 is a matrix protein of unknown function since, unlike other LTBPs, it does not form covalent complexes with latent TGF-beta. We have previously shown that LTBP-2 has widespread association with fibrillin-containing microfibrils in developing aorta and other tissues. We have now shown that full-length human recombinant LTBP-2 specifically binds to the amino-terminal region of fibrillin-1, but not to fibrillin-2, in solid phase assays and overlay blotting. The binding was enhanced by the inclusion of 2 mM Ca2+ ions in the assay buffer and abolished by 5 mM EDTA indicating that the interaction was directly or indirectly Ca2+ ion dependent. The K(d) for the interaction was calculated from the specific binding curve as 9.4 nM. A recombinant carboxyl-terminal fragment of LTBP-2 was shown to a) bind the amino-terminal fragment of fibrillin-1 and b) block completely the binding of full length LTBP-2 to fibrillin-1. This result indicates that the major fibrillin-1 binding site resides close to the carboxyl-terminus of LTBP-2. Further competitive binding studies showed that an analogous carboxyl terminal fragment of LTBP-1 was able to block the binding of LTBP-2 to fibrillin-1 and that the C-terminal fragment of LTBP-2 could block the interaction of the LTBP-1 fragment with the fibrillin. Thus the binding site for LTBP-2 on fibrillin-1 appears to be the same or in close proximity to that for LTBP-1. Immunohistochemical analysis of developing human aorta showed distinctive but extensively overlapping distributions for LTBPs-1 and -2. Both LTBPs showed extensive co-localization with fibrillin-1 and elastic lamellae but LTBP-2 had extensive signal throughout the medial layer whereas LTBP-1 showed strong localization only in the outer medial layer. The finding indicates that there is a possibility for LTBP-2 to compete with LTBP-1 for binding to fibrillin-containing microfibrils throughout the aortic wall but particularly in the outer medial region where the LTBP-1 is predominantly located. Overall, the results support the concept that that LTBP-2 may be an indirect negative modulator for storage of the large latent TGF-beta complex on microfibrils in aorta and other fibrillin-rich tissues.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.     

Apolipoprotein A-I directly interacts with extracellular domain 1 of human ABCA1

ATP-binding cassette transporter A1 (ABCA1) is critical for the generation of nascent high-density lipoprotein (HDL) and plays important roles in cholesterol homeostasis. ABCA1 has two large extracellular domains (ECDs), which may interact directly with apolipoprotein A-I (apoA-I). However, the molecular mechanisms underlying HDL formation and the importance of ABCA1–apoA-I interactions in HDL formation remain unclear. We investigated the ABCA1–apoA-I interaction in photo-activated crosslinking experiments using sulfo-SBED–labeled apoA-I. ApoA-I bound to cells expressing ABCA1, but not to untransfected cells or cells expressing non-functional ABCA1. Binding was inhibited by sulfo-SBED–labeled apoA-I, and crosslinking of sulfo-SBED–labeled apoA-I with ABCA1 was inhibited by non-labeled apoA-I, suggesting that sulfo-SBED–labeled apoA-I specifically binds and crosslinks with functional ABCA1. Proteolytic digestion of crosslinked ABCA1 revealed that apoA-I bound the N-terminal half of ABCA1, and that the first ECD of ABCA1 is an apoA-I binding site.

Abbreviations: ABC: ATP-binding cassette; apoA-I: apolipoprotein A-I; ATP: adenosine triphosphate; CHAPS: 3-(3-cholamidepropyl)dimethylammonio-1- propanesulphonate; DTT: dithiothreitol; ECD: extra cellular domain; EDTA: ethylenediaminetetraacetic acid; GFP: green fluorescent protein; HA: hemagglutinin; HDL: high density lipoprotein; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; sulfo-SBED: (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido] ethyl-1,3?-dithiopropionate; NHS-ester, N-hydroxysuccinimide-ester     

Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity

Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH₂-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.     

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.     

Human cellular prion protein interacts directly with clusterin protein

Prion protein is a glycosyl-phosphatidyl-inositol anchored glycoprotein localized on the surface and within a variety of cells. Its conformation change is thought to be essential for the proliferation of prion neurodegenerative diseases. Using the yeast two-hybrid assay we identified an interaction between prion protein and clusterin, a chaperone glycoprotein. This interaction was confirmed in a mammalian system by in vivo co-immunoprecipitation and in vitro by circular dichroism analysis. Through deletion mapping analysis we demonstrated that the alpha subunit, but not the beta subunit, of clusterin binds to prion and that the C-terminal 62 amino acid segment of the putative alpha helix region of clusterin is essential for the binding interaction. The full prion protein as well as the N-terminal section (aa 23-95) and C-terminal (aa 96-231) were shown to interact with clusterin. These findings provide new insights into the molecular mechanisms of interaction between prion and clusterin protein and contribute to the understanding of prion protein's physiological function.     

BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function

BRCA1 interacts in vivo with a novel protein, BACH1, a member of the DEAH helicase family. BACH1 binds directly to the BRCT repeats of BRCA1. A BACH1 derivative, bearing a mutation in a residue that was essential for catalytic function in other helicases, interfered with normal double-strand break repair in a manner that was dependent on its BRCA1 binding function. Thus, BACH1/BRCA1 complex formation contributes to a key BRCA1 activity. In addition, germline BACH1 mutations affecting the helicase domain were detected in two early-onset breast cancer patients and not in 200 matched controls. Thus, it is conceivable that, like BRCA1, BACH1 is a target of germline cancer-inducing mutations.     

Nephrocystin-1 interacts directly with Ack1 and is expressed in human collecting duct

Nephronophthisis is characterised by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end stage renal failure. Mutations in NPHP1 account for the underlying genetic defect in 25% of patients with nephronophthisis. Loss of urine concentration ability may be an early feature of nephronophthisis. Using yeast-2-library screening with the SH3 domain of nephrocystin-1 as bait, we identify Ack1 as a novel interaction partner. This interaction is confirmed using exogenous over-expression followed by co-immunoprecipitation. Ack1 is an activated Cdc42-associated kinase, and like nephrocystin-1, is a known interactor of p130Cas. Nephrocystin-1 partially colocalises with Ack1 at cell-cell contacts in IMCD3 cells. In human kidney, nephrocystin-1 expression is limited to cell-cell junctions in renal collecting duct cells. These data define Ack1 as a novel interaction partner of nephrocystin-1 and implicate cell-cell junctions and the renal collecting duct in the pathology of nephronophthisis.     

Menin interacts directly with the homeobox-containing protein Pem

Scalarane-type sesterterpenes, PHC-1-PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1-5 microg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells.     

U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression

Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5' splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5' splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and?an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.     

Caveolin-1 interacts directly with dynamin-2

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.     

A novel GTPase-activating protein for ARF6 directly interacts with clathrin and regulates clathrin-dependent endocytosis

ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.     

A型流感病毒PB1-F2蛋白与MOAP-1的相互作用

【目的】探讨A型流感病毒PB1-F2蛋白和人类凋亡调节因子1(MOAP-1)之间的相互作用。【方法】构建pACT2-MOAP-1重组质粒,与pGBKT7-PB1-F2质粒共转化酵母AH109,检测转化菌在四缺培养基的生长情况及β半乳糖苷酶报告基因的活性;利用GST pull-down和免疫共沉淀(Co-IP)技术进一步验证PB1-F2与宿主细胞蛋白MOAP-1的相互作用;通过过表达PB1-F2和MOAP-1,检测PB1-F2对MOAP-1蛋白表达水平的影响。【结果】酵母双杂交结果表明,PB1-F2和MOAP-1可以在酵母细胞内特异性结合。GST pull-down和Co-IP实验也进一步证实了这两种蛋白的相互作用,而且PB1-F2可上调外源MOAP-1的蛋白水平。【结论】流感病毒PB1-F2与MOAP-1存在相互作用,PB1-F2可能通过与MOAP-1的相互作用参与调控细胞生长及凋亡过程。     

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer’s disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.