C-terminal Coiled-coil Domain Research Articles

Effect of Oxidative Stress on Homer Scaffolding Proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

Structure and Dynamics of a Stabilized Coiled-Coil Domain in the P-TEFb Regulator Hexim1

The positive transcription elongation factor P-TEFb mediates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb is negatively regulated by the cellular protein Hexim1 (hexamethylene bisacetamide-inducible protein 1), which is highly conserved in higher eukaryotes. The C-terminal coiled-coil domain of Hexim1 recognizes the Cyclin T subunit of P-TEFb, whereas a central PYNT motif is required to inhibit the cyclin-dependent kinase Cdk9 by a yet unknown mechanism. Here, the crystal structure of the Cyclin T-binding domain (TBD) of human Hexim1 was determined at 2.1 Å resolution using a deletion mutant of three residues in its central stammer motif. The structure showed a continuous parallel coiled-coil domain of nine hepta-repeats with a preceding helix encompassing up to 15 residues. Two uncommon residues at heptad a positions in the N-terminal part of the coiled-coil structure, Lys284 and Tyr291, stabilize the preceding helix by a tight intermolecular hydrogen bond network with residues of the opposing chain. These interactions delineate a characteristic turn between both helices that is supposed to mediate binding to Cyclin T1. Stabilization of the coiled-coil domain by deletion of the stammer region was confirmed by NMR spectroscopic and backbone dynamic analyses analyzing wild-type TBD and three mutant variants. This study thus provides structural insights into the recognition of the regulator protein Hexim1 by P-TEFb and the modulation of coiled-coil dynamics by specific discontinuities.

The Structural Determinations of the Leucine Zipper Coiled-Coil Domains of the cGMP-Dependent Protein Kinase Iα and Its Interaction with the Myosin Binding Subunit of the Myosin Light Chains Phosphase

Physiologic relaxation of vascular smooth muscle is induced by the cyclic guanosine monophosphate (cGMP)- dependent protein kinase Iα enzyme (cGKIα), which activates myosin phosphatase (MLCP). This activation process is thought to occur through the interaction involving both N- and C-terminal leucine zipper coiled-coil (LZCC) domains of the kinase enzyme (cGKIα) with the myosin binding subunit (MBS) of MLCP. In this review, I summarize how to define the LZCC domains in both N-terminal cGKIα(1-59) and C-terminal MBS proteins using predictive and experimental methods, how to make a rapid and accurate structure determination of a cGKIα(1-59) molecule using NMR's residual dipolar coupling (RDC) measurements, and how to indentify the existence of a weak protein interaction between N-terminal LZCC domain (cGKIα(1-59)) and a LZCC domain (MBSCT42) within the C-terminal MBS. In addition, the location and orientation of the residues in LZCC proteins can be readily visualized using a novel diagram, the so-called "wenxiang diagram", which is more advantageous than traditional helical wheel diagrams in analyzing LZCC protein structures and their action mechanisms. Using the composed wenxiang diagrams, we have characterized the interaction between cGKIα(1- 59) and another LZCC molecule (MBSCT42), and deduced that the most affected residues of these two LZCC molecules might be at the positions d, a, e and g. These studies and findings are also covered in this review. It is intriguing to see that the successful incorporation of wenxiang diagrams and NMR spectroscopy in the LZCC structural and functional studies may provide some insights into protein-protein interaction mechanisms.

Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled‐coil domain

Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region.

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

A Single Amino Acid Residue Constitutes the Third Dimerization Domain Essential for the Assembly and Function of the Tetrameric Polycystin-2 (TRPP2) Channel

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a nonselective calcium channel. PC2 has been found to form oligomers in native tissues, suggesting that similar to other TRP channels, it may form functional homo- or heterotetramers with other TRP subunits. We have recently demonstrated that the homodimerization of PC2 is mediated by both N-terminal and C-terminal domains, and it is known that PC2 can heterodimerize with PC1, TRPC1, and TRPV4. In this paper, we report that a single cysteine residue, Cys632, mutated in a known PKD2 pedigree, constitutes the third dimerization domain for PC2. PC2 truncation mutants lacking both N and C termini could still dimerize under nonreducing conditions. Mutation of Cys632 alone abolished dimerization in these mutants, indicating that it was the critical residue mediating disulfide bond formation between PC2 monomers. Co-expression of C632A PC2 mutants with wild-type PC2 channels reduced ATP-sensitive endoplasmic reticulum Ca2+ release in HEK293 cells. The combination of C632A and mutations disrupting the C-terminal coiled-coil domain (Val846, Ile853, Ile860, Leu867 or 4M) nearly abolished dimer formation and ATP-dependent Ca2+ release. However, unlike the 4M PC2 mutant, a C632A mutant could still heterodimerize with polycystin-1 (PC1). Our results indicate that PC2 homodimerization is regulated by three distinct domains and that these events regulate formation of the tetrameric PC2 channel.

HZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10-interaction domain

Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein-dynactin and Mad1-Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.

Structure-Function Analysis of the Anopheles gambiae LRIM1/APL1C Complex and its Interaction with Complement C3-Like Protein TEP1

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.

Occludin is essential for tumor necrosis factor (TNF)‐induced intestinal epithelial tight junction (TJ) disruption

TNF decreases intestinal epithelial TJ barrier function by a myosin light chain kinase- (MLCK-) dependent process. Although endocytosis of occludin following TNF-induced MLCK activation is required for barrier loss, specific contributions of occludin are poorly defined. Our aim was to define the role of occludin domains in TNF-induced TJ disruption. Stable occludin knockdown (KD) decreased transepithelial resistance (TER) of Caco-2 monolayers to 59 ± 7 % of wild type (WT). Analysis of cation and FITC-dextran flux showed that this was primarily due to increased permeability to large solutes; 3kDa FITC-dextran flux was 474 ± 52 % of control. Expression and trafficking of other TJ proteins were unaffected by occludin KD. TNF decreased TER of IFNγ-primed WT monolayers by 14 ± 1 % after 4 hrs, but TER of occludin KD monolayers was unchanged, despite increased MLC phosphorylation. Stable reexpression of WT occludin, but not a mutant lacking the C-terminal coiled-coil domain, resensitized occludin KD monolayers to TNF-induced barrier loss. This was not due to defective trafficking, as the occludin deletion mutant was efficiently delivered to the TJ. Thus, occludin contributes to TNF-induced TJ barrier loss by a mechanism that involves the C-terminal coiled-coil domain. This suggests that protein interactions mediated by the coiled-coil domain are required for TNF-induced barrier loss. Supported by NIH and CCFA.

Far3p domains involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in budding yeast

Far3p (factor arrest), a protein that interacts with Far7-11p, is required for the pheromone-mediated cell cycle arrest in G1 phase. We used a combination of computational and experimental strategies to identify the Far3p self-association, to map the Far3p domains that interact with Far3p itself and with other Far proteins, and to reveal the importance of the two coiled-coil motifs of Far3p in the integrity and function of the Far complex. We show that Far3p self-associates through its central region and its C-terminal coiled-coil domain, that the amino acid 61-100 region of Far3p interacts with Far7p, and that the Far3p N-terminal coiled-coil domain interacts with Far9p and Far10p. Mutation of the N-terminal coiled coil disrupts the interactions of Far3p with Far9p and Far10p, and mutation of the C-terminal domain weakens the Far3p self-interaction. Although the N- and C-terminal coiled-coil mutants reserve some of the interactions with itself and some other Far proteins, both mutants are defective in the pheromone-mediated G1 arrest, indicating that both coiled-coil motifs of Far3p are essential for the integrity and the function of the Far complex.

A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae.

The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.

SZIP, an Alternative Splice Variant of ZIP, Antagonizes Transcription Repression and Growth Inhibition by ZIP

Recently, we reported a novel transcriptional repressor, ZIP (for zinc finger and G-patch domain-containing), which recruits the Mi-2/NuRD (nucleosome remodeling and deacetylase) complex and represses the expression of epidermal growth factor receptor (EGFR). In doing so, ZIP inhibits cell proliferation and suppresses breast carcinogenesis. Here, we report the cloning and the characterization of an alternatively spliced isoform of ZIP, sZIP. sZIP is an N-terminal truncated form of ZIP, lacking the zinc finger but retaining part of the G-patch domain and C-terminal coiled-coil domain of ZIP. We showed that sZIP could interact with the NuRD complex but lost its DNA-binding capacity. We demonstrated that sZIP antagonizes the transcription repression by ZIP by competing for the binding of the NuRD complex and that sZIP alleviates the growth inhibitory effect of ZIP on hepatocarcinoma cells through attenuating the transcriptional repression of EGFR. Our data provide a finely tuned mechanism for EGFR regulation and add another player for transcription repression.

A polycystin-2 (TRPP2) dimerization domain essential for the function of heteromeric polycystin complexes

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Earlier work has shown that PC1 and PC2 assemble into a polycystin complex implicated in kidney morphogenesis. PC2 also assembles into homomers of uncertain functional significance. However, little is known about the molecular mechanisms that direct polycystin complex assembly and specify its functions. We have identified a coiled coil in the C-terminus of PC2 that functions as a homodimerization domain essential for PC1 binding but not for its self-oligomerization. Dimerization-defective PC2 mutants were unable to reconstitute PC1/PC2 complexes either at the plasma membrane (PM) or at PM-endoplasmic reticulum (ER) junctions but could still function as ER Ca2+-release channels. Expression of dimerization-defective PC2 mutants in zebrafish resulted in a cystic phenotype but had lesser effects on organ laterality. We conclude that C-terminal dimerization of PC2 specifies the formation of polycystin complexes but not formation of ER-localized PC2 channels. Mutations that affect PC2 C-terminal homo- and heteromerization are the likely molecular basis of cyst formation in ADPKD.

Rab13 Regulates Neurite Outgrowth in PC12 Cells through Its Effector Protein, JRAB/MICAL-L2

Neurite outgrowth is the first step in the processes of neuronal differentiation and regeneration and leads to synaptic polarization and plasticity. Rab13 small G protein shows an increased mRNA expression level during neuronal regeneration; it is therefore thought to be involved in this process. We previously identified JRAB (junctional Rab13-binding protein)/MICAL-L2 (molecules interacting with CasL-like 2) as a novel Rab13 effector protein. Here, we show that Rab13 regulates neurite outgrowth in the rat pheochromocytoma cell line PC12 through an interaction with JRAB/MICAL-L2. The expression of JRAB/MICAL-L2 alone inhibits neurite outgrowth, whereas coexpression of the dominant active form of Rab13 rescues this effect. We also demonstrate an intramolecular interaction between the N-terminal calponin-homology (CH) and LIM domains of JRAB/MICAL-L2 and the C-terminal coiled-coil domain. Finally, we show that the binding of Rab13 to JRAB/MICAL-L2 stimulates the interaction of JRAB/MICAL-L2 with actinin-4, an actin-binding protein, which localizes to the cell body and the tips of the neurites in PC12 cells. These results suggest that Rab13 and JRAB/MICAL-L2 may act to transfer actinin-4 from the cell body to the tips of neurites, where actinin-4 is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth.

Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins

BackgroundThe discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, Anopheles gambiae, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR) containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM) proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains.ResultsThe identification and characterisation of genes related to LRIM1 and APL1C revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of LRIM1 and APL1C identified more than 20 LRIM-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: An. gambiae, Aedes aegypti, and Culex quinquefasciatus. Comparative sequence analyses revealed that this family of mosquito LRIM-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains.ConclusionsThe evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation of LRIM protein complexes or mediate interactions with other immune proteins. The conserved LRIM cysteine residue patterns are likely to be important for structural fold stability and the formation of protein complexes. These sequence-structure-function relations of mosquito LRIMs will serve to guide the experimental elucidation of their molecular roles in mosquito immunity.

Immunodetection and Oligomerisation of TRPM1

To study expression and assembly of TRPM1 channels we cloned seven full lengths TRPM1 cDNAs from mouse eye which represent splice variants and encode proteins of 1462, 1506, 1512, 1578, 1584, 1622 and 1628 amino acid residues. These TRPM1 protein variants vary in their N terminus, in a putative extracellular loop and the predicted pore region. For expression analyses we prepared polyclonal and monoclonal antibodies directed against various antigenic epitopes of TRPM1. All antibodies detect proteins of the size expected for TRPM1 in the eye, in lung and skeletal muscle. They immunoprecipitate the TRPM1 protein from mouse retina and specifically label postsynaptic dendrites of bipolar cells adjacent to the ribbon synapse. All TRPM1 proteins contain a coiled-coil domain in their C termini and isolated fragments comprising this coiled-coil domain have been shown to form dimers (1). We show that after disruption of this coiled-coil domain by site directed mutagenesis the full-length TRPM1 proteins still assemble to dimers and higher molecular protein complexes and interact with each other, apparently by additional intermolecular interactions of TRPM1 sequences within the N-terminus. Similarily, a related TRPM protein, TRPM4, still forms dimers after destruction of a C-terminal coiled-coil domain, most probably via interactions of N-terminal protein sequences. 1 Tsuruda PR, Julius D, Minor DL Jr (2007) Neuron 51, 201-212.

Ubiquitin-like Sequence in ASK1 Plays Critical Roles in the Recognition and Stabilization by USP9X and Oxidative Stress-Induced Cell Death

Ubiquitination is an important posttranslational modification that regulates various cellular processes, including signal transduction. However, physiological roles of ubiquitination in the regulation of MAPK pathways are poorly understood. Here, we identified the deubiquitinating enzyme USP9X as a binding partner of ASK1 that mediates oxidative stress-induced cell death through activation of the JNK and p38 MAPK pathways. In the recognition of ubiquitin by deubiquitinating enzymes, the importance of a tandem glycine-glycine sequence in the ubiquitin C terminus has been suggested. Interestingly, ASK1 contains six amino acids identical to the ubiquitin C terminus (LRLRGG), and the GG sequence of ASK1 was required for the USP9X-ASK1 interaction. We also found that USP9X interacted with oxidative stress-activated ASK1 and prevented it from undergoing ubiquitin-dependent degradation. In USP9X-deficient cells, oxidative stress-induced JNK activation and subsequent cell death were reduced. These results demonstrate that USP9X-dependent stabilization of activated ASK1 plays a crucial role in oxidative stress-induced cell death.

Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

Dynamins at a glance

The superfamily of dynamins includes classical dynamins and dynamin-related proteins. Classical dynamins are proteins that share sequence similarity with the first described dynamin, which is a large GTPase with five characteristic domains. Dynamin-related proteins differ from classical dynamins in

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.