The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs

Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery. Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA). GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein. Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes. Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin. These motifs are found within different helices within the three-helix GAT subdomain. When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.     

Crystal structure of the human GGA1 GAT domain

GGAs are a family of vesicle-coating regulatory proteins that function in intracellular protein transport. A GGA molecule contains four domains, each mediating interaction with other proteins in carrying out intracellular transport. The GAT domain of GGAs has been identified as the structural entity that binds membrane-bound ARF, a molecular switch regulating vesicle-coat assembly. It also directly interacts with rabaptin5, an essential component of endosome fusion. A 2.8 A resolution crystal structure of the human GGA1 GAT domain is reported here. The GAT domain contains four helices and has an elongated shape with the longest dimension exceeding 80 A. Its longest helix is involved in two structural motifs: an N-terminal helix-loop-helix motif and a C-terminal three-helix bundle. The N-terminal motif harbors the most conservative amino acid sequence in the GGA GAT domains. Within this conserved region, a cluster of residues previously implicated in ARF binding forms a hydrophobic surface patch, which is likely to be the ARF-binding site. In addition, a structure-based mutagenesis-biochemical analysis demonstrates that the C-terminal three-helix bundle of this GAT domain is responsible for the rabaptin5 binding. These structural characteristics are consistent with a model supporting multiple functional roles for the GAT domain.     

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.     

The interaction of the human GGA1 GAT domain with rabaptin-5 is mediated by residues on its three-helix bundle

GGA proteins regulate clathrin-coated vesicle trafficking by interacting with multiple proteins during vesicle assembly. As part of this process, the GAT domain of GGA is known to interact with both ARF and Rabaptin-5. Particularly, the GAT domains of GGA1 and -2, but not of GGA3, specifically bind with a coiled-coil region of Rabaptin-5. Rabaptin-5 interacts with Rab5 and is an essential component of the fusion machinery for targeting endocytic vesicles to early endosomes. The recently determined crystal structure of the GGA1 GAT domain has provided insights into its interactions with partner proteins. Here, we describe mutagenesis studies on the GAT-Rabaptin-5 interaction. The results demonstrate that a hydrophobic surface patch on the C-terminal three-helix bundle motif of the GAT domain is directly involved in Rabaptin-5 binding. A GGA3-like mutation, N284S, in this Rabaptin-5 binding patch of GGA1 led to a reduced level of Rabaptin-5 binding. Furthermore, a reversed mutation, S293N, in GGA3 partially establishes Rabaptin-5 binding ability in its GAT domain. These results provide a structural explanation for the binding affinity difference among GGA proteins. The current results also suggest that the binding of GAT to Rabaptin-5 is independent of its interaction with ARF.     

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.     

The ubiquitin ligase gene (WWP1) is responsible for the chicken muscular dystrophy

Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.     

泛素、泛素链和蛋白质泛素化研究进展

蛋白质泛素化是以泛素单体和泛素链作为信号分子,共价修饰细胞内其他蛋白质的一种翻译后修饰形式。不同蛋白质底物、同一底物的不同氨基酸修饰位点以及同一位点上泛素链连接方式的不同均可导致细胞效应的差异。蛋白质泛素化在真核细胞内广泛存在,除了介导蛋白质的26S蛋白酶体降解途径之外,还广泛参与了基因转录、蛋白质翻译、信号传导、细胞周期控制以及生长发育等几乎所有的生命活动过程。泛素链的形成及其修饰过程的任何失调均可导致生物体内环境的紊乱,从而产生严重的疾病。文中结合实验室研究,综述了泛素的发现历史、基因特点、晶体结构,特别是泛素链的组装过程、结构、功能以及与人类相关疾病关系的新进展,可为这些疾病的治疗靶点和药物靶标的研究提供思路。     

SQSTM1/p62 (sequestosome 1) senses cellular ubiquitin stress through E2-mediated ubiquitination

The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub⁺ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub⁺ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub⁺ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.     

The domain responsible for sphingomyelin synthase (SMS) activity

Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS.     

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.     

The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2

ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation were resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity.     

Perturbed heme binding is responsible for the blistering phenotype associated with mutations in the Caenorhabditis elegans dual oxidase 1 (DUOX1) peroxidase domain

Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.     

Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain

Cdc48 (p97/VCP) is an AAA-ATPase molecular chaperone whose cellular functions are facilitated by its interaction with ubiquitin binding cofactors (e.g., Npl4-Ufd1 and Shp1). Several studies have shown that Saccharomyces cerevisiae Doa1 (Ufd3/Zzz4) and its mammalian homologue, PLAA, interact with Cdc48. However, the function of this interaction has not been determined, nor has a physiological link between these proteins been demonstrated. Herein, we demonstrate that Cdc48 interacts directly with the C-terminal PUL domain of Doa1. We find that Doa1 possesses a novel ubiquitin binding domain (we propose the name PFU domain, for PLAA family ubiquitin binding domain), which appears to be necessary for Doa1 function. Our data suggest that the PUL and PFU domains of Doa1 promote the formation of a Doa1-Cdc48-ubiquitin ternary complex, potentially allowing for the recruitment of ubiquitinated proteins to Cdc48. DOA1 and CDC48 mutations are epistatic, suggesting that their interaction is physiologically relevant. Lastly, we provide evidence of functional conservation within the PLAA family by showing that a human-yeast chimera binds to ubiquitin and complements doa1Delta phenotypes in yeast. Combined, our data suggest that Doa1 plays a physiological role as a ubiquitin binding cofactor of Cdc48 and that human PLAA may play an analogous role via its interaction with p97/VCP.     

The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.     

Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1

Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases

The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Here, we show that Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promoted ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. We suggest that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases.