Mammalian tolloid-like 1 binds procollagen C-proteinase enhancer protein 1 and differs from bone morphogenetic protein 1 in the functional roles of homologous protein domains

Bone morphogenetic protein 1 (BMP1) is the prototype of a subgroup of metalloproteinases with manifold roles in morphogenesis. Four mammalian subgroup members exist, including BMP1 and mammalian Tolloid-like 1 (mTLL1). Subgroup members have a conserved protein domain structure: an NH2-terminal astacin-like protease domain, followed by a fixed order of CUB and epidermal growth factor-like protein-protein interaction motifs. Previous structure/function studies have documented those BMP1 protein domains necessary for secretion, and activity against various substrates. Here we demonstrate that, in contradiction to previous reports, the most NH2-terminal CUB domain (CUB1) is not required for BMP1 secretion nor is the next CUB domain (CUB2) required for enzymatic activity. The same is true for mTLL1. In fact, secreted protease domains of BMP1 and mTLL1, devoid of CUB or epidermal growth factor-like domains, have procollagen C-proteinase (pCP) activity and activity for biosynthetic processing of biglycan, the latter with kinetics superior to those of the full-length proteins. Structure-function analyses herein also suggest differences in the functional roles played by some of the homologous domains in BMP1 and mTLL1. Surprisingly, although BMP1 has long been known to be Ca2+-dependent, a property previously assumed to apply to all members of the subgroup, mTLL1 is demonstrated to be independent of Ca2 levels in its ability to cleave some, but not all, substrates. We also show that pCP activities of only versions of BMP1 and mTLL1 with intact COOH termini are enhanced by the procollagen C-proteinase enhancer 1 (PCOLCE1) and that mTLL1 binds PCOLCE1, thus suggesting reappraisal of the accepted paradigm for how PCOLCE1 enhances pCP activities.     

Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

Stabilization of RNA strands in protein synthesis by type I procollagen C-proteinase enhancer protein, a potential RNA-binding protein, in hepatic stellate cells.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.     

Cloning of two new splice variants of Siglec-10 and mapping of the interaction between Siglec-10 and SHP-1

Using a three-hybrid strategy in yeast, we have cloned a new splice variant of Siglec-10, called Siglec-10 Sv3. This splice variant lacks part of exon 3, but keeps the reading frame, as well as the crucial regions for interaction with Sias and the motifs for intracellular signaling. The expression of Siglec-10 Sv3 in T- and B-cells was detected by RT-PCR. Moreover, cDNA of another new splicing form of Siglec-10, named Siglec-10 Sv4, was identified by RT-PCR. One common characteristic of all Siglec-10 splice forms (except for Siglec-10 Sv2) is their cytoplasmic tail with two ITIMs and one CD150-like sequence. We confirmed the recruitment of SHP-1 to the Siglec-10 cytoplasmic tail by Western blot analysis and demonstrated that this interaction depends on tyrosine phosphorylation. Mutational analyses showed that ITIM Y609 of Siglec-10 and the N-terminal SH2 domain of SHP-1 play a pivotal role in the interaction between Siglec-10 and SHP-1. Finally, we demonstrated that Siglec-10 was not able to bind SAP/SH2d1A, indicating that the so-called CD150-like motif in Siglec-10 might be a docking site for other signal transduction mediators.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.     

Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356: evidence for protein-protein interaction of two subdomains

The structure of the crystallisation domain, SAN, of the S(A)-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S(A)-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or within regions of sequence variation in SAN (amino acid position 7, 45, 114, 125, 193), or in the cell wall-binding domain (position 345) could form crystalline sheets, whereas insertions in conserved regions or in regions with predicted secondary structure elements (positions 30, 67, 88 and 156) destroyed this capacity. FACscan analysis of L.acidophilus synthesising three crystallising and one non-crystallising S(A)-protein c-myc (19 amino acid residues) insertion mutant was performed with c-myc antibodies. Fluorescence was most pronounced for insertions at positions 125 and 156, less for position 45 and severely reduced for position 7. By cytometric flow sorting a transformant harbouring the mutant S(A)-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant S(A)-protein only. PCR analysis of the transformant grown in the absence of selection pressure indicated that the mutant allele was stably integrated in the chromosome. Proteolytic treatment of S(A)-protein indicated that only sites near the middle of SAN are susceptible, although potential cleavage sites are present through the entire molecule. Expression in E.coli of DNA sequences encoding the two halves of SAN yielded peptides that could oligomerize. Our results indicate that SAN consists of a approximately 12kDa N and a approximately 18kDa C-terminal subdomain linked by a surface exposed loop. The capacity of S(A)-protein of L.acidophilus to present epitopes, up to approximately 19 amino acid residues in length, at the bacterial surface in a genetically stable form, makes the system, in principle, suitable for application as an oral delivery vehicle.     

Identification and expression of a novel type I procollagen C-proteinase enhancer protein gene from the glaucoma candidate region on 3q21-q24

A novel human Type I procollagen C-proteinase enhancer protein-like gene, PCOLCE2, was identified by sequencing an EST in the primary open-angle glaucoma (POAG) region on 3q21. The total cDNA encoded a 415-amino-acid protein that has 43% identity to the Type I procollagen C-proteinase enhancer protein (PCOLCE1). PCOLCE2 contains two CUB domains, which are thought to be involved in protein-protein interactions, and an NTR module. PCOLCE2 message is expressed in the trabecular meshwork, lungs, heart, brain, liver, skeletal muscle, kidney, pancreas, and placenta as a 2-kb message. PCOLCE2, a 52-kDa protein, is expressed in the trabecular meshwork. A novel gene, PCOLCE2, has been identified and characterized. Based upon its homology with collagen-binding proteins, its expression in the trabecular meshwork, and its chromosome location, PCOLCE2 is a candidate gene for GLC1C. However, no coding sequence mutations were detected in PCOLCE2 in a POAG patient from the GLC1C family.     

PCOLCE2 encodes a functional procollagen C-proteinase enhancer (PCPE2) that is a collagen-binding protein differing in distribution of expression and post-translational modification from the previously described PCPE1

The procollagen COOH-terminal proteinase enhancer (PCPE) is a glycoprotein that binds the COOH-terminal propeptide of type I procollagen and potentiates its cleavage by procollagen C-proteinases, such as bone morphogenetic protein-1 (BMP-1). Recently, sequencing of a human expressed sequence tag, which maps near the primary open angle glaucoma region on chromosome 3q21, showed it to encode a novel protein with only 43% identity with PCPE but with a similar domain structure. Here we show this novel protein to be a functional procollagen COOH-terminal proteinase enhancer with activity comparable with that of PCPE and thus propose the designations PCPE2 and PCPE1, respectively. PCPE2 is shown to have a much more limited distribution of expression than does PCPE1, with strong expression primarily in nonossified cartilage in developing tissues and at high levels in the adult heart. PCPE2 is shown to be a glycoprotein that differs markedly in the nature of its glycosylation from that of PCPE1. PCPE2 is also shown to have markedly stronger affinity for heparin than PCPE1, which may account for higher affinities for cell layers. Unexpectedly, both PCPE1 and PCPE2 were found to be collagen-binding proteins, capable of binding at multiple sites on the triple helical portions of fibrillar collagens and also capable of competing for such binding with procollagen C-proteinases. The latter observations may provide insights into the ways PCPEs affect the kinetics of the C-proteinase reaction and into the physical interactions that occur between procollagen C-proteinases and their substrates.     

Mechanism of differences in pathogenicity between two variants of a laboratory strain of herpes simplex virus type 1

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.     

LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads

Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.     

Negative regulation of Epstein-Barr virus latent membrane protein 1-mediated functions by the bone morphogenetic protein receptor IA-binding protein,BRAM1

The latent membrane protein 1 (LMP1) of Epstein-Barr virus causes cellular transformation and activates several intracellular signals, including NF-kappaB and c-Jun N-terminal kinase. Using yeast two-hybrid screening with the LMP1 C-terminal sequence as bait, we demonstrate that BRAM1 (bone morphogenetic protein receptor-associated molecule 1) is an LMP1-interacting protein. BRAM1 associates with LMP1, both in vitro and in vivo, as revealed by confocal microscopy, glutathione S-transferase pull-down, and co-immunoprecipitation assays. This association mainly involves the C-terminal half of BRAM1 comprising the MYND domain and the CTAR2 region of LMP1, which is critical in LMP1-mediated signaling pathways. We show that BRAM1 interferes with LMP1-mediated NF-kappaB activation but not the JNK signaling pathway. Because the CTAR2 region interacts with the tumor necrosis factor (TNF-alpha receptor-associated death domain protein, it is interesting to find that BRAM1 also interferes with NF-kappaB activation mediated by TNF-alpha. BRAM1 interferes LMP1-mediated and TNF-alpha-induced NF-kappaB activation by targeting IkappaBalpha molecules. Moreover, BRAM1 inhibits the resistance of LMP1-expressing cells to TNF-alpha-induced cytotoxicity. We therefore propose that the BRAM1 molecule associates with LMP1 and functions as a negative regulator of LMP1-mediated biological functions.     

Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (approximately 6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via "structural mimicry" due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.     

The residues determining differences in ion affinities among the alternative splice variants F, A, and B of the mammalian renal Na-K-Cl cotransporter (NKCC2)

Three alternatively spliced variants of the renal Na-K-Cl cotransporter (NKCC2) are found in distinct regions of the thick ascending limb of the mammalian kidney; these variants mediate Na(+)K(+)2Cl(-) transport with different ion affinities. Here, we examine the specific residues involved in the variant-specific affinity differences, utilizing a mutagenic approach to change the NKCC2B variant into the A or F variant, with functional expression in Xenopus oocytes. The splice region contains the second transmembrane domain (TM2) and the putative intracellular loop (ICL1) connecting TM2 and TM3. It is found that the B variant is functionally changed to the F variant by replacement of six residues, half of the effect brought about by three TM2 residues and half by three ICL1 residues. The involvement of the ICL1 residues strongly suggests that this region of ICL1 may actually be part of a membrane-embedded domain. Changing six residues is also sufficient to bring about the smaller functional change from the B to the A variant; three residues in TM2 appear to be primarily responsible, two of which correspond to residues involved in the B-to-F changes. A B-variant mutation reported in a mild case of Bartter disease was found to render the cotransporter inactive. These results identify the combination of amino acid variations responsible for the differences among the three splice variants of NKCC2, and they support a model in which a reentrant loop following TM2 contributes to the chloride binding and translocation domains.     

The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.     

Evidence for functional conservation of a mammalian homologue of the light-responsive plant protein COP1.

Identified in Arabidopsis as a repressor of light-regulated development, the COP1 (constitutively photomorphogenic 1) protein is characterized by a RING-finger motif and a WD40 repeat domain [1]. The subcellular localization of COP1 is light-dependent. COP1 acts within the nucleus to repress photomorphogenic development, but light inactivates COP1 and diminishes its nuclear abundance [2]. Here, we report the identification of a mammalian COP1 homologue that contains all the structural features present in Arabidopsis COP1 (AtCOP1). When expressed in plant cells, a fusion protein comprising mammalian COP1 and beta-glucuronidase (GUS) responded to light by changing its subcellular localization pattern in a manner similar to AtCOP1. Whereas the mammalian COP1 was unable to rescue the defects of Arabidopsis cop1 mutants, expression of the amino-terminal half of mammalian COP1 in Arabidopsis interfered with endogenous COP1 function, resulting in a hyperphotomorphogenic phenotype. Therefore, the regulatory modules in COP1 proteins that are responsible for the signal-dependent subcellular localization are functionally conserved between higher plants and mammals, suggesting that mammalian COP1 may share a common mode of action with its plant counterpart in regulating development and cellular signaling.