Acylation of proteins with myristic acid occurs cotranslationally

Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC3H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [3H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [3H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [3H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification.     

Road to ruin: targeting proteins for degradation in the endoplasmic reticulum

Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.     

Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly

The 70-kilodalton family of heat shock proteins (Hsp 70) has been implicated in posttranslational protein assembly and translocation. Binding of cytosolic forms of Hsp 70 (Hsp 72,73) with nascent proteins in the normal cell was investigated and found to be transient and adenosine triphosphate (ATP)-dependent. Interaction of Hsp 72,73 with newly synthesized proteins appeared to occur cotranslationally, because nascent polypeptides released prematurely from polysomes in vivo can be isolated in a complex with Hsp 72,73. Moreover, isolation of polysomes from short-term [35S]Met-labeled cells (pulsed) revealed that Hsp 72,73 associated with nascent polypeptide chains. In cells experiencing stress, newly synthesized proteins coimmunoprecipitated with Hsp 72,73; however, in contrast to normal cells, interaction with Hsp 72,73 was not transient. A model consistent with these data suggests that under normal growth conditions, cytosolic Hsp 72,73 interact transiently with nascent polypeptides to facilitate proper folding, and that metabolic stress interferes with these events.     

ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.     

Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis

The protein modifier ubiquitin is a signal for proteasome-mediated degradation in eukaryotes. Proteasome-bearing prokaryotes have been thought to degrade proteins via a ubiquitin-independent pathway. We have identified a prokaryotic ubiquitin-like protein, Pup (Rv2111c), which was specifically conjugated to proteasome substrates in the pathogen Mycobacterium tuberculosis. Pupylation occurred on lysines and required proteasome accessory factor A (PafA). In a pafA mutant, pupylated proteins were absent and substrates accumulated, thereby connecting pupylation with degradation. Although analogous to ubiquitylation, pupylation appears to proceed by a different chemistry. Thus, like eukaryotes, bacteria may use a small-protein modifier to control protein stability.     

In vivo half-life of a protein is a function of its amino-terminal residue

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.     

Biochemistry. Ubiquitination--more than two to tango

The ubiquitin pathway in the cell is an elegant system for targeting unwanted proteins for degradation. Three enzymes, E1, E2, and E3, are responsible for attaching the ubiquitin tag to proteins destined to be chopped up. In their Perspective, Joazeiro and Hunter discuss new structural findings that reveal the part played by an E3 called c-Cbl in this ubiquitinating process.     

U-box蛋白质的结构及其功能研究进展

泛素系统是选择性降解细胞内蛋白质的重要系统之一,U-box蛋白质是此系统中决定底物特异性识别的一种新型E3蛋白质,部分U-box蛋白质属于泛素链聚集因子-E4。U-box结构域大约由70个氨基酸残基构成,在酵母、植物和动物等真核生物中保守存在,但植物中的数目远多于动物中。该蛋白质在细胞内异常蛋白质的降解及质量控制方面具有重要的作用,了解U-box蛋白质的功能对疾病的发生控制机理有重要意义。     

A specificity-enhancing factor for the ClpXP degradation machine

Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.     

泛素/26S蛋白酶体途径在水稻中的生物学功能研究进展

生物体内的蛋白质降解方式有两种,一种不需要能量,一种需要能量。而泛素/26S蛋白酶体途径便是目前已知的依赖ATP、高效、有高度选择性的蛋白降解途径。它介导了真核生物中80%~85%的蛋白质降解,几乎参与到植物生长发育的各个环节,是植物体内蛋白高效专一降解最重要、最精细的调控机制之一。概述了泛素蛋白酶体途径,重点阐述了泛素结合酶E2和泛素连接酶E3的蛋白结构及其在水稻生长发育、激素信号传导、生物和非生物胁迫响应中的生物学功能及机制,并对进一步研究进行了展望,将有助于揭示泛素/26S蛋白酶体途径在水稻生长发育中的精细调控过程,并为水稻抗逆育种提供了指导和借鉴。     

Degradation of proteins with acetylated amino termini by the ubiquitin system

A free NH2-terminal group has been previously shown to be an obligatory signal for recognition and subsequent degradation of proteins in a partially fractionated and reconstituted ubiquitin proteolytic system. Naturally occurring proteins with acetylated NH2-termini--most cellular proteins fall in this category--were not degraded by this system. Other studies have suggested that the identity of the NH2-terminal residue is important in determining the metabolic stability of a protein in vivo (N-end rule). Whole reticulocyte lysate and antibodies directed against the ubiquitin-activating enzyme (E1) have now been used to show that such acetylated proteins are degraded in a ubiquitin-dependent mode. Although fractionation of lysate does not affect its proteolytic activity toward substrates with free NH2-termini, it completely abolishes the activity toward the blocked substrates, indicating that an important component of the system was either removed or inactivated during fractionation. An NH2-terminal "unblocking" activity that removes the blocking group, thus exposing a free NH2-terminus for recognition according to the N-end rule, does not seem to participate in this pathway. Incubation of whole lysate with labeled histone H2A results in the formation of multiple ubiquitin conjugates. In contrast, the fractionated system is devoid of any significant conjugating activity. These results suggest that a novel conjugating enzyme (possibly a ubiquitin-protein ligase) may be responsible for the degradation of these acetylated proteins by recognizing structural features of the substrate that are downstream and distinct from the NH2-terminal residue.     

Biochemistry. All in the ubiquitin family

The job of a protein can be altered by addition of molecules such as ubiquitin or the related ubiquitin-like modifiers, which bring about changes in the protein's localization, conformation, or its interactions with other proteins. In a comprehensive Perspective, Hochstrasser brings us up to date with the many new members of the ubiquitin modifier family and their multitudinous and diverse protein targets.     

泛素化修饰在RLR信号通路中的研究进展

病毒入侵后被细胞的模式识别受体RIG-I样受体(RIG-I-like receptor, RLR)识别从而启动抗病毒RLR信号通路的激活,先天免疫反应的异常激活将导致慢性炎症和免疫器官损伤,甚至引起自身免疫性疾病。为了防止抗病毒信号过早激活或过度激活,机体建立了完善的调节系统防止信号传导过程发生紊乱。蛋白的翻译后修饰(Post-translational modification, PTM)是调节模式识别受体及其下游信号蛋白稳定性和活性的关键机制,而泛素化(Ubiquitination, UB)作为蛋白质翻译后修饰的重要部分在抗病毒信号通路中被广泛研究。其中K48和K63连接的泛素化最为常见,通过K48连接的泛素链能够引起靶蛋白通过蛋白酶体途径降解,而K63连接的泛素链能够促进蛋白激活和细胞信号转导。RIG-I、MAVS、TBK1以及TRAF家族相关蛋白作为RLR通路的信号传递分子,其蛋白的泛素化修饰也成为研究的重点。本文讨论了K48和K63泛素化在抗病毒免疫信号通路中的研究进展,特别是RIG-I样受体引发的信号传导途径中蛋白的泛素化修饰。     

Protein synthesis upon acute nutrient restriction relies on proteasome function

The mechanisms that protect mammalian cells against amino acid deprivation are only partially understood. We found that during an acute decrease in external amino acid supply, before up-regulation of the autophagosomal-lysosomal pathway, efficient translation was ensured by proteasomal protein degradation. Amino acids for the synthesis of new proteins were supplied by the degradation of preexisting proteins, whereas nascent and newly formed polypeptides remained largely protected from proteolysis. Proteasome inhibition during nutrient deprivation caused rapid amino acid depletion and marked impairment of translation. Thus, the proteasome plays a crucial role in cell survival after acute disruption of amino acid supply.     

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Plant development: regulation by protein degradation

Many aspects of eukaryotic development depend on regulated protein degradation by the ubiquitin-proteasome pathway. This highly conserved pathway promotes covalent attachment of ubiquitin to protein substrates through the sequential action of three enzymes called a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). Most ubiquitinated proteins are then targeted for degradation by the 26S proteasome. Recent studies have also shown that the ubiquitin-related protein RUB/Nedd8 and the proteasome-related COP9 signalosome complex cooperate with the ubiquitin-proteasome pathway to promote protein degradation. Most of these components are conserved in all three eukaryotic kingdoms. However, the known targets of the pathway in plants, and the developmental processes they regulate, are specific to the plant kingdom.     

Regulatory role of SGT1 in early R gene-mediated plant defenses

Animal SGT1 is a component of Skp1-Cullin-F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.