New roles for ubiquitin in the assembly and function of neuronal circuits

A series of recent papers highlight a prominent role for ubiquitin in the formation and function of neural circuits. These new results focus attention on the molecular remodeling that occurs at various decision points in the life of growth cones and synapses.     

Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.     

The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity

Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein-protein interactions with the E2.     

Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.     

Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (Delta YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, Delta YRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.     

Inferring protein function by domain context similarities in protein-protein interaction networks

Background  

Genome sequencing projects generate massive amounts of sequence data but there are still many proteins whose functions remain unknown. The availability of large scale protein-protein interaction data sets makes it possible to develop new function prediction methods based on protein-protein interaction (PPI) networks. Although several existing methods combine multiple information resources, there is no study that integrates protein domain information and PPI networks to predict protein functions.     

Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain

During clathrin‐mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin‐binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile α‐motif (SAM) domain and shows a propensity to oligomerize. By co‐immunoprecipitation, Sla1p binds to clathrin and self‐associates in vivo. Mutations in the clathrin‐binding motif that abolish clathrin binding and structure‐based mutations in SHD2 that impede self‐association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin‐mediated endocytosis.     

Myosin filament assembly requires a cluster of four positive residues located in the rod domain

Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins.

Structured summary of protein interactions

WT LMMbinds to WT LMM by surface plasmon resonance (View Interaction)WT LMMbinds to CT2 LMM by surface plasmon resonance (View Interaction)WT LMMbinds to Alanine mutant LMM by surface plasmon resonance (View Interaction)WT LMM and WT LMMbind by light scattering (View Interaction)Alanine mutant LMM and Alanine mutant LMMbind by light scattering (View Interaction)WT LMM and Alanine mutant LMMbind by light scattering (View Interaction)     

Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1 CA

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion as well as viral infectivity. Previous in vivo experiments generated two N-terminal domain charge change mutants (E45A and E128A/R132A) that showed an increase in stability of the viral core. This increase in core stability resulted in decreased infectivity, suggesting the need for a delicate balance of favorable and unfavorable interactions to both allow assembly and facilitate uncoating following infection. Purified CA protein can be triggered to assemble into tubelike structures through the use of a high salt buffer system. The requirement for high salt suggests the need to overcome charge/charge repulsion between subunits. The mutations mentioned above lie within a highly charged region of the N-terminal domain of CA, away from any of the proposed protein/protein interaction sites. We constructed a number of charge mutants in this region (E45A, E45K, E128A, R132A, E128A/R132A, K131A, and K131E) and evaluated their effect on protein stability in addition to their effect on the rate of CA assembly. We find that the mutations alter the rate of assembly of CA without significantly changing the stability of the CA monomer. The changes in rate for the mutants studied are found to be due to varying degrees of electrostatic repulsion between the subunits of each mutant.     

Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

Trafficking of Lyn through the Golgi caveolin involves the charged residues on alphaE and alphaI helices in the kinase domain

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced "closed conformation" but not by kinase inactivation. Four residues (Asp346 and Glu353 on alphaE helix, and Asp498 and Asp499 on alphaI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an "open conformation," are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.     

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear'' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain'' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains.     

A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis

Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis. This endocytic process requires AP180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles. The highly conserved 33 kDa amino-terminal domain of AP180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of AP180. The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides. The finding that the clathrin-box motif is mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.     

TFEB,a novel mTORC1 effector implicated in lysosome biogenesis,endocytosis and autophagy

Comment on: Peña-Llopis S, et al. EMBO J 2011; 30:3242-58.     

RIP death domain structural interactions implicated in TNF-mediated proliferation and survival

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).     

Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix

Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.     

ATP-dependent hexameric assembly of the heat shock protein Hsp101 involves multiple interaction domains and a functional C-proximal nucleotide-binding domain

Members of the Hsp100 family of heat stress proteins are present in species throughout the bacterial, plant, and fungal kingdoms. Most Hsp100 proteins are composed of five domains that include two nucleotide-binding domains required for their ATP-dependent oligomerization. Mutations within the first but not the second nucleotide-binding site disrupt self-assembly of bacterial Hsp100, whereas the reverse is true for yeast Hsp104. We have examined the functional requirements for oligomerization of plant Hsp101 and have found that Hsp101 resembles Hsp104 in that it assembles into a hexameric complex in an ATP-dependent manner. Self-assembly of Hsp101 involves at least three distinct interaction domains located in the N-proximal domain and in the first and second nucleotide-binding domains. The interaction domain in the second nucleotide-binding domain included the Walker A motif, and mutations within this element disrupted self-assembly of Hsp101. In contrast, mutations affecting conserved residues of the Walker A motif within the first nucleotide-binding site did not affect self-assembly. No interaction between Hsp101 and Hsp104 was observed. These results suggest that plant Hsp101 self-assembly involves multiple evolutionarily diverged interaction domains as well as an evolutionarily conserved requirement for a functional C-proximal nucleotide-binding site.     

Juxtamembrane basic residues in glycoprotein Ibbeta cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex

Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149-154 in the cytoplasmic domain of the GPIbbeta subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbbeta by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbbeta cytoplasmic domain in biosynthesis of the GPIb-IX complex.