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PubMed2004

A clinical, genetic and candidate gene study of Silver syndrome, a complicated form of hereditary spastic paraplegia.

Silver syndrome (SS) is a complicated form of hereditary spastic paraplegia associated with distal wasting of the small muscles of the hands. We have previously described a large kindred with SS and mapped a genetic locus (SPG17) to chromosome 11q12-q14. In the current study we analyse the clinical phenotype and perform linkage analysis in three new SS families. In addition we analyse candidate genes mapping to the SS locus (SPG17). Clinical assessments were performed on 25 (15 affected) individuals from each family in which SS segregates with variable clinical expression. Neurophysiological studies, performed in the index case of two families, suggested anterior horn cell or nerve root involvement. Linkage analysis using microsatellite markers mapping to the SPG17 locus was performed and only one of the three families had a microsatellite segregation pattern compatible with linkage. Candidate genes mapping to the SS critical region were analysed in this and one other SPG17-linked family. Mutation analysis of genes encoding calpain 1 ( CAPN1), copper chaperone for superoxide dismutase ( CCS), ADP ribosylation factor-like 2 ( ARL2), LOC120664, a putative homologue of atlastin ( ATLSTL-1) and sorting nexin 15 ( SNX15) failed to identify any disease-specific mutations. SS therefore exhibits both clinical and genetic heterogeneity and the SPG17 locus may account for a significant proportion of SS mutations in the UK.

Adolescent↗
PubMed2006

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor.

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.

Animals↗
PubMed2004

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting.

The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the five dopamine receptors (D1-D5) were expressed as glutathione S-transferase (GST) fusion proteins and studied for their interaction with ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) and N-ethylmaleimide-sensitive factor (NSF), which are known to be involved in post-endocytic recycling of receptors back to the plasma membrane, and with sorting nexin 1 (SNX1), known to be involved in targeting receptors to lysosomal degradation. EBP50 did not bind any of the dopamine receptor tails. NSF bound strongly to D1 and D5 and only weakly to D2, D3 and D4. However, SNX1 clearly distinguished between D1 and D5, as only D5 bound strongly to this protein. This report shows that there are distinct interaction patterns for NSF and SNX1 to the various dopamine receptor subtypes.

Animals↗
PubMed2003

cDNA cloning and characterization of a novel SNX gene differentially expressed in previtellogenic oocytes of gibel carp.

To understand the molecular events governing fish oogenesis, a multiple technique was used to identify the genes differentially expressed at different phases during fish oogenesis. This technique is a combination of suppression subtractive hybridization, SMART cDNA synthesis and RACE-PCR. Here we report the cDNA cloning and expression characterization of a novel SNX gene based on its differential transcription between previtellogenic and fully mature oocytes in naturally gynogenetic gibel carp. First, a cDNA fragment selectively expressed in previtellogenic oocytes was identified and used to screen a SMART cDNA library prepared from the same mRNA sample by RACE-PCR for cloning fully length cDNA. The full length cDNA was 1392-bp long and coded for a novel SNX protein with 225 amino acids. The 5' UTR had 72 bp and 3' UTR had 642 bp. Unlike most of maternal genes that are transcribed after vitellogenesis and stored in oocytes, this gene is expressed at a higher level in the previtellogenic oocytes and at a much lower level in fully matured oocytes. However, RT-PCR analysis of tissues showed it was ubiquitous transcription. The novel gene is named fish sorting nexin (fSNX), because it contains a conserved PX domain. The fact which major expression of the gene occurs in the previtellogenic oocytes suggests that it might have an important function in the oogenesis.

Amino Acid Sequence↗
PubMed2001

SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P.

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

Androstadienes↗
PubMed2005

Vps29 has a phosphoesterase fold that acts as a protein interaction scaffold for retromer assembly.

The retromer complex is responsible for the retrieval of mannose 6-phosphate receptors from the endosomal system to the Golgi. Here we present the crystal structure of the mammalian retromer subunit mVps29 and show that it has structural similarity to divalent metal-containing phosphoesterases. mVps29 can coordinate metals in a similar manner but has no detectable phosphoesterase activity in vitro, suggesting a unique specificity or function. The mVps29 and mVps26 subunits bind independently to mVps35 and together form a high-affinity heterotrimeric subcomplex. Mutagenesis reveals the structural basis for the interaction of mVps29 with mVps35 and subsequent association with endosomal membranes in vivo. A conserved hydrophobic surface distinct from the primary Vps35p binding site mediates assembly of the Vps29p-Vps26p-Vps35p subcomplex with sorting nexins in yeast, and mutation of either site results in a defect in retromer-dependent membrane trafficking.

Amino Acid Sequence↗
PubMed2004

The BAR-domain family of proteins: a case of bending and binding?

BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinbeta family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome.

We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.

ADAM Proteins↗
PubMed2004

Interaction of activated Cdc42-associated tyrosine kinase ACK2 with HSP90.

ACK2 (activated Cdc42-associated tyrosine kinase 2) is a specific downstream effector for Cdc42, a member of the Rho family of small G-proteins. ACK2 interacts with clathrin, an endocytic vesicle coating protein, and SH3PX1, a sorting nexin, and is involved in clathrin-mediated endocytosis. While searching for proteins that interact with ACK2, we found that HSP90 (heat-shock protein 90) binds to ACK2. Analysis of a series of truncation mutants of ACK2 has defined the regions within the kinase domain of ACK2 that are required for binding to HSP90. The binding of HSP90 to ACK2 is blocked upon treatment with geldanamycin, an HSP90-specific ATPase inhibitor, and is required for the in vivo kinase activity of ACK2 and its association with Cdc42. Overall, our data suggest a novel mechanism of regulation in which HSP90 serves as a regulatory component in an ACK2 functional complex and plays a role in sustaining its kinase activity.

Adenosine Triphosphatases↗
PubMed2003

Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate.

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.

Amino Acid Sequence↗
PubMed2005

Crystal structure of human vacuolar protein sorting protein 29 reveals a phosphodiesterase/nuclease-like fold and two protein-protein interaction sites.

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a "retromer complex." In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Angstroms resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered alpha-beta-beta-alpha sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two protein-protein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.

Amino Acid Sequence↗
PubMed2000

Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.

Amino Acid Sequence↗
PubMed2005

Control of Ste6 recycling by ubiquitination in the early endocytic pathway in yeast.

We present evidence that ubiquitination controls sorting of the ABC-transporter Ste6 in the early endocytic pathway. The intracellular distribution of Ste6 variants with reduced ubiquitination was examined. In contrast to wild-type Ste6, which was mainly localized to internal structures, these variants accumulated at the cell surface in a polar manner. When endocytic recycling was blocked by Ypt6 inactivation, the ubiquitination deficient variants were trapped inside the cell. This indicates that the polar distribution is maintained dynamically through endocytic recycling and localized exocytosis ("kinetic polarization"). Ste6 does not appear to recycle through late endosomes, because recycling was not blocked in class E vps (vacuolar protein sorting) mutants (Deltavps4, Deltavps27), which are affected in late endosome function and in the retromer mutant Deltavps35. Instead, recycling was partially affected in the sorting nexin mutant Deltasnx4, which serves as an indication that Ste6 recycles through early endosomes. Enhanced recycling of wild-type Ste6 was observed in class D vps mutants (Deltapep12, Deltavps8, and Deltavps21). The identification of putative recycling signals in Ste6 suggests that recycling is a signal-mediated process. Endocytic recycling and localized exocytosis could be important for Ste6 polarization during the mating process.

ATP-Binding Cassette Transporters↗
PubMed2004

Novel interaction partners of the TPR/MET tyrosine kinase.

A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

Animals↗
PubMed2005

A novel mammalian retromer component, Vps26B.

The mammalian retromer protein complex, which consists of three proteins--Vps26, Vps29, and Vps35--in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

Amino Acid Sequence↗
PubMed2003

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

ADP-Ribosylation Factors↗
PubMed2006

Btn2, a Hook1 ortholog and potential Batten disease-related protein, mediates late endosome-Golgi protein sorting in yeast.

BTN2 gene expression in the yeast Saccharomyces cerevisiae is up-regulated in response to the deletion of BTN1, which encodes the ortholog of a human Batten disease protein. We isolated Btn2 as a Snc1 v-SNARE binding protein using the two-hybrid assay and examined its role in intracellular protein trafficking. We show that Btn2 is an ortholog of the Drosophila and mammalian Hook1 proteins that interact with SNAREs, cargo proteins, and coat components involved in endosome-Golgi protein sorting. By immunoprecipitation, it was found that Btn2 bound the yeast endocytic SNARE complex (e.g., Snc1 and Snc2 [Snc1/2], Tlg1, Tlg2, and Vti1), the Snx4 sorting nexin, and retromer (e.g., Vps26 and Vps35). In in vitro binding assays, recombinant His(6)-tagged Btn2 bound glutathione S-transferase (GST)-Snc1 and GST-Vps26. Btn2-green fluorescent protein and Btn2-red fluorescent protein colocalize with Tlg2, Snx4, and Vps27 to a compartment adjacent to the vacuole that corresponds to a late endosome. The deletion of BTN2 blocks Yif1 retrieval back to the Golgi apparatus, while the localization of Ste2, Fur4, Snc1, Vps10, carboxypeptidases Y (CPY) and S (CPS), Sed5, and Sec7 is unaltered in btn2Delta cells. Yif1 delivery to the vacuole was observed in other late endosome-Golgi trafficking mutants, including ypt6Delta, snx4Delta, and vps26Delta cells. Thus, Btn2 facilitates specific protein retrieval from a late endosome to the Golgi apparatus, a process which may be adversely affected in patients with Batten disease.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Profiling of differentially expressed genes in wound margin biopsies of horses using suppression subtractive hybridization.

Disturbed gene expression may disrupt the normal process of repair and lead to pathological situations resulting in excessive scarring. To prevent and treat impaired healing, it is necessary to first define baseline gene expression during normal repair. The objective of this study was to compare gene expression in normal intact skin (IS) and wound margin (WM) biopsies using suppression subtractive hybridization (SSH) to identify genes differentially expressed during wound repair in horses. Tissue samples included both normal IS and biopsies from 7-day-old wounds. IS cDNAs were subtracted from WM cDNAs to establish a subtracted (WM-IS) cDNA library; 226 nonredundant cDNAs were identified. Detection of genes previously shown to be expressed 7 days after trauma, including the pro-alpha(2)-chain of type 1 pro-collagen (COL1A2), annexin A(2), the pro-alpha(3)-chain of type 6 pro-collagen, beta-actin, fibroblast growth factor 7, laminin receptor 1, matrix metalloproteinase 1 (MMP1), secreted protein acidic cystein rich, and tissue inhibitor of metalloproteinase 2, supported the validity of the experimental design. A RT-PCR assay confirmed an increase or induction of the cDNAs of specific genes (COL1A2, MMP1, dermatan sulfate proteoglycan 2, cluster differentiation 68, cluster differentiation 163, and disintegrin and metalloproteinase domain 9) within wound biopsies. Among these, COL1A2 and MMP1 had previously been documented in horses; 68.8% of the cDNAs had not previously been attributed a role during wound repair, of which spermidine/spermine-N-acetyltransferase, serin proteinase inhibitor B10, and sorting nexin 9 were highly expressed and whose known functions in other processes made them potential candidates in regulating the proliferative response to wounding. In conclusion, we identified novel genes that are differentially expressed in equine wound biopsies and that may modulate repair. Future experiments must correlate changes in mRNA levels for precise molecules with spatiotemporal protein expression within tissues.

Animals↗