Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8

Post-translational modification by ubiquitin-like proteins (Ublps) is an essential cellular regulatory mechanism. The Ublp NEDD8 regulates cell division, signalling and embryogenesis. Ublps are conjugated to their targets by the sequential action of E1, E2 and often E3 enzymes. Each Ublp has a dedicated E1, or activating enzyme, that initiates its conjugation cascade. First, E1 associates with the Ublp and catalyses adenylation of the carboxy terminus of the Ublp. Second, E1 forms a thioester between its catalytic cysteine and the Ublp. Next, E1 is loaded with a second Ublp molecule, adenylating the C terminus of this second Ublp while still carrying the first thioester-bound Ublp. Last, E1 binds E2 and promotes Ublp transfer to the catalytic cysteine of E2. We report here the structure and mutational analysis of human APPBP1-UBA3, the heterodimeric E1 enzyme for NEDD8 (ref. 11). Each E1 activity is specified by a domain: an adenylation domain resembling bacterial adenylating enzymes, an E1-specific domain organized around the catalytic cysteine, and a domain involved in E2 recognition resembling ubiquitin. The domains are arranged around two clefts that coordinate protein and nucleotide binding so that each of E1's reactions drives the next, in an assembly-line fashion.     

类泛素化修饰Neddylation在心脏生物学中的作用

Neural precursor cell-expressed developmentally downregulated 8(NEDD8)是类泛素蛋白家族的一员,其结构与泛素相似,通过E1激活酶、E2结合酶和E3连接酶等酶促级联反应对蛋白进行翻译后修饰,这一过程即为neddylation。Neddylation异常已被证实与癌症、神经退行性疾病和先天性心脏病等多种疾病密切相关。近年来,neddylation和deneddylation(底物上的NEDD8在deneddylation酶的作用下被去除,称为deneddylation)在心血管系统中的作用备受关注,本文将主要阐述neddylation的生物学过程及其在心脏生物学中的作用。     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

Loss of autophagy in the central nervous system causes neurodegeneration in mice

Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy--the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover--in the protein quality-control process. However, little is known about the precise roles of autophagy in neurons. Here we report that loss of Atg7 (autophagy-related 7), a gene essential for autophagy, leads to neurodegeneration. We found that mice lacking Atg7 specifically in the central nervous system showed behavioural defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices. Notably, polyubiquitinated proteins accumulated in autophagy-deficient neurons as inclusion bodies, which increased in size and number with ageing. There was, however, no obvious alteration in proteasome function. Our results indicate that autophagy is essential for the survival of neural cells, and that impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.     

An interferon-induced cellular enzyme is incorporated into virions

The mechanisms by which interferon inhibits viral growth are only partially understood. Several enzymatic activities increase in cells shortly after treatment with interferon. One of these enzymes, oligo-isoadenylate synthetase, synthesizes (2'-5') isoadenylate oligomers which strongly stimulate the activity of a cellular ribonuclease, RNase F (ref. 7). Interferon also significantly increases the activity of a protein kinase which phosphorylates the initiation factor eIF-2 and can inhibit in vitro protein synthesis. Such interferon-induced enzymes, which affect RNA and protein metabolism, might be responsible for many of its effects on viruses. Indeed, inhibition of viral protein and RNA synthesis appears to have a major role in the antiviral state. We have now investigated possible interactions of the two enzymes with viral constituents during the course of infection and found that in two different membrane-coated RNA viruses, vesicular stomatitis virus (VSV) and Moloney murine leukaemia virus (M-MuLV), there is an accumulation of the 2'-5') oligo-isoadenylate synthetase (E) in the virions. Most of the enzyme is bound to the virion ribonucleoprotein core. The incorporation of E into the virions suggests a direct involvement of the enzyme in regulation of virus functions.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Detection of refolding conformers of complement protein C9 during insertion into membranes

Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.     

Trembler mouse carries a point mutation in a myelin gene.

The autosomal dominant trembler mutation (Tr), maps to mouse chromosome 11 (ref. 2) and manifests as a Schwann-cell defect characterized by severe hypomyelination and continuing Schwann-cell proliferation throughout life. Affected animals move clumsily and develop tremor and transient seizures at a young age. We have recently described a potentially growth-regulating myelin protein, peripheral myelin protein-22 (PMP-22; refs 7, 8), which is expressed by Schwann cells and found in peripheral myelin. We now report the assignment of the gene for PMP-22 to mouse chromosome 11. Cloning and sequencing of PMP-22 complementary DNAs from inbred Tr mice reveals a point mutation that substitutes an aspartic acid residue for a glycine in a putative membrane-associated domain of the PMP-22 protein. Our results identify the PMP-22 gene as a likely candidate for the mouse trembler locus and will encourage the search for mutations in the corresponding human gene in pedigrees with hypertrophic neuropathies such as Charcot-Marie-Tooth and Dejerine-Sottas diseases (hereditary motor and sensory neuropathies I and III).     

几乎单DD-群

研究几乎单的DD-群, 证明了几乎单群G不是一个DD-群, 如果G不是下列群中的一个: 1) 散在单群M₂₂, J₂, Co₁, Fi'₂₄, McL, Th, B, 以及M₁₂或者J₂的自同构群; 2) 交错群A₅, A₆, A₇, A₉, A₁₀, A₁₆, S₅, Aut(A₆), S₈, S₁₀, 或者A_n(62≤n≤205); 3) L₃(2), Aut(L₃(3)), 或者L₂(q), 其中q=4, 5, 7, 9, 11。     

Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.     

Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.     

Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex

Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.     

石斑鱼肌肉氨基酸组成的初步研究

本文测定了花点石斑鱼和青石斑鱼肌肉中18种氨基酸的含量。测定结果表明:石斑鱼肌肉中氨基酸含量较高,花点石斑鱼肌肉中氨基酸总含量为764.98mg/g,青石斑鱼的为743.78mg/g,这两种石斑鱼肌肉的氨基酸总舍量中,人体的8种必需氨基酸含量之和分别为40.87%和41,99%;谷氨酸、天门冬氨酸,精氨酸、丙氨酸和甘氨酸等5种氨基酸系的鲜味物质的含量之和分别为45.03%和44.37%.石斑鱼肌肉所含必需氨基酸的比值与人体所需氨基酸的组成模式比较接近.所以,对人体来说,石斑鱼肌肉是一种营养价值较高的鲜美的蛋白源.最后,本文提出了石斑鱼必需氨基酸的组成模式。     

Induction of autophagy and inhibition of tumorigenesis by beclin 1

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.     

A new protease required for cell-cycle progression in yeast

In eukaryotes, protein function can be modulated by ligation to ubiquitin or to ubiquitin-like proteins (Ubl proteins). The vertebrate Ubl protein SUMO-1 is only 18% identical to ubiquitin but is 48% identical to the yeast protein Smt3. Both SUMO-1 and Smt3 are ligated to cellular proteins, and protein conjugation to SUMO-1/Smt3 is involved in many physiological processes. It remained unknown, however, whether deconjugation of SUMO-1/Smt3 from proteins is also essential. Here we describe a yeast Ubl-specific protease, Ulp1, which cleaves proteins from Smt3 and SUMO-1 but not from ubiquitin. Ulp1 is unrelated to any known deubiquitinating enzyme but shows distant similarity to certain viral proteases, indicating the existence of a widely conserved protease fold. Proteins related to Ulp1 are present in many organisms, including several human pathogens. The pattern of Smt3-coupled proteins in yeast changes markedly throughout the cell cycle, and specific conjugates accumulate in ulp1 mutants. Ulp1 has several functions, including an essential role in the G2/M phase of the cell cycle.