RESUMO
Protein-protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indanos/farmacologia , Compostos Organometálicos/farmacologia , Quinonas/farmacologia , Zinco/farmacologia , Relação Dose-Resposta a Droga , Humanos , Indanos/química , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Ligação Proteica/efeitos dos fármacos , Quinonas/química , Relação Estrutura-Atividade , Zinco/químicaRESUMO
α-Helices are common secondary structural elements forming key parts of the large, generally featureless interfacial regions of many therapeutically-relevant protein-protein interactions (PPIs). The rational design of helix mimetics is an appealing small-molecule strategy for the mediation of aberrant PPIs, however the first generation of scaffolds presented a relatively small number of residues on a single recognition surface. Increasingly, helices involved in PPIs are found to have more complex binding modes, utilizing two or three recognition surfaces, or binding with extended points of contact. To address these unmet needs the design and synthesis of new generations of multi-sided, extended, and supersecondary structures are underway.
Assuntos
Biomimética , Desenho de Fármacos , Animais , Sítios de Ligação , Biomimética/tendências , Calmodulina/química , Humanos , Estrutura Secundária de ProteínaRESUMO
Cellular processes such as transcription and DNA repair may be regulated through diverse mechanisms, including RNA synthesis, protein synthesis, posttranslational modification and protein degradation. The 26S proteasome, which is responsible for degrading a broad spectrum of proteins, has been shown to interact with several nucleotide excision repair proteins, including xeroderma pigmentosum B protein (XPB), Rad4, and Rad23. Rad4 and Rad23 form a complex that binds preferentially to UV-damaged DNA. The 26S proteasome may regulate repair by degrading DNA repair proteins after repair is completed or, alternatively, the proteasome may act as a molecular chaperone to promote disassembly of the repair complex. In either case, the interaction between the proteasome and nucleotide excision repair depends on proteins like Rad23 that bind ubiquitin-conjugated proteins and the proteasome. While the iteration between Rad4 and Rad23 is well established, it will be interesting to determine what other proteins are regulated in a Rad23-dependent manner.
RESUMO
Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multi-ubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substrate-linked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Códon , DNA/metabolismo , Enzimas Reparadoras do DNA , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Delta was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Histonas/metabolismo , Humanos , Especificidade por Substrato , Enzimas de Conjugação de UbiquitinaRESUMO
The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.
Assuntos
Ligases/metabolismo , Complexos Multienzimáticos , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Ligação Competitiva , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Temperatura Alta , Ligases/genética , Mutação , Oligopeptídeos , Peptídeo Hidrolases/genética , Peptídeos/genética , Peptídeos/metabolismo , Enzimas de Conjugação de Ubiquitina , Leveduras/genética , Leveduras/metabolismoRESUMO
Regulation of protein expression can be achieved through destruction of proteins by the 26S: proteasome. Cellular processes that are regulated by proteolysis include cell cycle progression, stress responses and differentiation. Several nucleotide excision repair proteins in yeast and humans, such as Rad23, Rad4 and XPB, have been shown to co-purify with Cim3 and Cim5, AAA ATPases of the 19S: proteasome regulatory subunit. However, it has not been determined if nucleotide excision repair is regulated through protein destruction. We measured nucleotide excision repair in yeast mutants that are defective in proteasome function and found that the repair of the transcribed and non-transcribed strands of an RNA polymerase II-transcribed reporter gene was increased in the absence of proteasome function. Additionally, overexpression of the Rad4 repair protein, which is bound to the repair/proteolytic factor Rad23, conferred higher rates of nucleotide excision repair. Based on our data we suggest that a protein (or proteins) involved in nucleotide excision repair or in regulation of repair is degraded by the 26S proteasome. We propose that decreased proteasome function enables increased DNA repair, due to the transient accumulation of a specific repair factor, perhaps Rad4.
Assuntos
Reparo do DNA/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA , Genoma Fúngico , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Dímeros de Pirimidina/metabolismo , Proteínas de Schizosaccharomyces pombe , Transglutaminases , Leveduras/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , Adutos de DNA/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Cinética , Mutação/genética , Mutação/efeitos da radiação , Peptídeo Hidrolases/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/efeitos da radiação , Temperatura , Transcrição Gênica/genética , Raios Ultravioleta , Leveduras/enzimologia , Leveduras/crescimento & desenvolvimento , Leveduras/efeitos da radiaçãoRESUMO
Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Canavanina/farmacologia , Ciclo Celular , Temperatura Baixa , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Essenciais , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Teste de Complementação Genética , Peptídeo Hidrolases/genética , Fenótipo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Tempo , Ubiquitinas/metabolismo , Raios UltravioletaRESUMO
The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes. Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes. The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes. We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's. Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function. However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature. The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway. We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase). The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal.
Assuntos
Ligases/química , Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina , Substituição de Aminoácidos , Dicroísmo Circular , Clonagem Molecular/métodos , Cisteína , Estabilidade Enzimática , Temperatura Alta , Cinética , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , TermodinâmicaAssuntos
Cisteína Endopeptidases/química , Complexos Multienzimáticos/química , Saccharomyces cerevisiae/enzimologia , Animais , Cisteína Endopeptidases/genética , Humanos , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Saccharomyces cerevisiae/genética , Terminologia como AssuntoRESUMO
BACKGROUND: Gpa1 is the alpha subunit of the yeast G-protein that regulates signal transduction during mating. The stability of Galpha/Gpa1 is influenced by the ubiquitin-dependent N-end rule pathway, suggesting that the regulation of G alpha levels may be important for effective mating response and recovery. RESULTS: The G alpha sequences that confer sensitivity to degradation by the N-end rule pathway were identified. The insertion of this degradation signal (G1-Deg) into the ordinarily stable Gpa2 protein conferred proteolytic targeting. We examined G alpha degradation under different conditions and found that it was efficiently degraded in haploid and diploid cells, but was stable if it was synthesized prior to expression of the N-end rule pathway. Interestingly, a specific mutation in G alpha that is believed to promote the GTP-bound form (N388K) caused accelerated degradation. CONCLUSION: A region encompassing a putative effector-binding domain (G1-Deg) is required for G alpha degradation via the N-end rule pathway. Our studies have shown that G alpha is susceptible to proteolysis soon after synthesis. These results are in agreement with the idea that G alpha is more unstable in the GTP-bound form, which is the predominant state of monomeric/free G alpha soon after synthesis. It is likely that the signal transduced by Gbetagamma can be regulated by adjusting the levels of G alpha through proteolysis.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase , Proteínas Heterotriméricas de Ligação ao GTP , Peptídeo Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Diploide , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Haploidia , Feromônios , Biossíntese de Proteínas , Saccharomyces cerevisiae , Deleção de Sequência , Transdução de Sinais , Especificidade por Substrato , Fatores de TempoRESUMO
The discovery of protein ubiquitination in a broad range of organisms and experimental settings has raised the need for a straightforward way to characterize the mechanism of substrate targeting, using purified components. The mechanism of ubiquitin conjugation to proteins has been extensively studied and is mediated by a family of evolutionarily conserved proteins. We have used previously described expression systems to purify the relevant targeting components of the ubiquitin system. These methods yielded substantial amounts of highly purified and catalytically active enzymes that permitted their use in reconstituting protein ubiquitination. We monitored ubiquitination reactions with 32P-ubiquitin rather than 125I-ubiquitin. This advance makes the procedure accessible to a broader range of experimentalists, since it eliminates the additional concerns involved in handling 125I-isotope. Furthermore, the strategies described here can be used to investigate the effects of specific mutations introduced into ubiquitin or the targeting components (E1, Ubc/E2, and E3) of this pathway.
Assuntos
Ligases/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Bovinos , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Clonagem Molecular/métodos , Sequência Conservada , Primers do DNA , Glutationa Transferase/metabolismo , Radioisótopos do Iodo , Ligases/isolamento & purificação , Radioisótopos de Fósforo , Reação em Cadeia da Polimerase/métodos , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinas/químicaRESUMO
Rad23 is an evolutionarily conserved protein that is important for nucleotide excision repair. A regulatory role has been proposed for Rad23 because rad23 mutants are sensitive to ultraviolet light but are still capable of incising damaged DNA. Here we show that Rad23 interacts with the 26S proteasome through an amino-terminal ubiquitin-like domain (UbL[R23]). The carboxy terminus of Rad23 binds to the Rad4 DNA repair protein and creates a link between the DNA repair and proteasome pathways. The ultraviolet sensitivity caused by deletion of the UbL(R23) domain may therefore arise from its inability to interact with the proteasome. The fusion proteins glutathione S-transferase (GST)-Rad23 and Rad4-haemagglutinin (HA), and the proteasome subunits Cim3 and Cim5, cofractionate through consecutive chromatography steps. The ubiquitin-like domain of human Rad23 (UbL[HRB]) also interacts with the human proteasome. These results demonstrate that ubiquitin-like domains (UbLs) represent a new class of proteasome-interacting motifs.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Transglutaminases , Ubiquitinas/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutationa Transferase/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
OBJECTIVE: To examine the status of trauma system development and key structural and operational characteristics of these systems. DESIGN AND SETTING: National survey of trauma systems with enabling state statute, regulation, or executive orders and for which designated trauma centers were present. PARTICIPANTS: Trauma system administrators and directors of 37 state and regional organizations that had legal authority to administer trauma systems, which represented a response rate of 90.2%. MAIN OUTCOME MEASURES: Trauma system components that had been implemented or were under development. RESULTS: From 1988 to 1993, the number of states meeting one set of criteria for a complete trauma system criteria increased from two to five. The most common deficiency in establishing trauma systems was failure to limit the number of designated trauma centers based on community need. Although most existing trauma systems have developed formal processes for designating trauma centers, prehospital triage protocols to allow hospital bypass, and centralized trauma registries, several systems lack standardized policies for interhospital transfer and systemwide evaluation. CONCLUSION: State and regional organizations have accomplished a great deal but still have substantial work ahead in developing comprehensive trauma systems. Research is needed to better understand the relationship between trauma volume and outcomes of care as well as the impact of trauma system structure and operational characteristics on care delivery. Improved measures of patient outcome are also needed so that effective system evaluation can take place.
Assuntos
Programas Médicos Regionais/organização & administração , Centros de Traumatologia/organização & administração , Centros de Traumatologia/estatística & dados numéricos , Protocolos Clínicos , Coleta de Dados , Interpretação Estatística de Dados , Geografia , Humanos , Modelos Organizacionais , Transferência de Pacientes/normas , Programas Médicos Regionais/legislação & jurisprudência , Programas Médicos Regionais/estatística & dados numéricos , Triagem/normas , Estados UnidosRESUMO
The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Overexpression of targeting components of the N-end rule pathway in Saccharomyces cerevisiae inhibited the growth of haploid but not diploid cells. This ploidy-dependent toxicity was shown to result from enhanced degradation of Gpa1, the alpha subunit (G alpha) of a heterotrimeric guanine nucleotide-binding protein (G protein) that regulates cell differentiation in response to mating pheromones. Sst2, a protein whose absence renders cells hypersensitive to pheromone, was essential for degradation of G alpha but not other N-end rule substrates, suggesting the involvement of an indirect, or trans-, targeting mechanism. G alpha degradation by the N-end rule pathway adds another regulatory dimension to the multitude of signaling functions mediated by G proteins.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases , Divisão Celular , Dipeptídeos/farmacologia , Guanosina Trifosfato/metabolismo , Meia-Vida , Haploidia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética , Enzimas de Conjugação de UbiquitinaRESUMO
Purpose. This study assesses buddy support in a community-based, minimal-contact smoking cessation program. Design. Telephone interviews with participants (n=641, response=74%) before and after (end-of-program, n=1,023, response=83%; three months n=757, response=74%; six months, n=859, response=84%; and 12 months, n=713, response=70%) intervention provided the data to be analyzed. Setting. The Chicago metropolitan area was the setting. Subjects. Subjects were a random sample of registrants for the intervention program. Intervention. A self-help smoking cessation program was used, which included a manual and complementary televised segments. Engaging a buddy was optional. Measures. Background and psychosocial characteristics of participants, characteristics of buddies, program compliance, and smoking behavior were the measures used. Results. Almost one third (30.3%) engaged a buddy. Those most likely to engage a buddy were female (33.4%), younger than 30 (37.2%), educated beyond high school (33.4%), highly determined to quit (41.8%), and more likely to need help from others (39.8%). More than half of the buddies were from outside the participant's household (55.1%), and more than half were nonsmokers (60.9%). Having a buddy was associated positively with manual use (gamma=.38), viewing televised segments (gamma=.23), recalling manual segments (gamma=.33), and recalling televised segments (gamma=.26). Among those who read the manual least, having a buddy was associated with viewing televised segments (gamma=.26, p less than .05) and with end-of-program quitting (16.8% vs. 9.8%, p less than .05). Having a buddy also was associated with higher abstinence through 12 months (5.8% vs. 2.7%, p=.013). Among those with lower determination, the end-of-program quit rate was more than three times greater (p=.013) for those with a buddy (16.1%) than without a buddy (5.2%). Participants whose buddy was their spouse or partner were more likely to quit at end-of-program (29.1% vs. 18.4%, p=.031). Conclusions. Buddy support should be promoted as an adjunct to minimal-contact smoking cessation programs. Impact of buddy support might be improved by guiding participants in choosing a buddy.
Assuntos
Abandono do Hábito de Fumar , Apoio Social , Adulto , Idoso , Feminino , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Avaliação de Programas e Projetos de Saúde , Resultado do TratamentoRESUMO
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, substrates of the N-end rule pathway are targeted for degradation by a complex that includes the 225-kDa N-recognin, encoded by UBR1, and the 20-kDa ubiquitin-conjugating enzyme encoded by UBC2. We report that both physical stability and functional activity of the N-recognin.Ubc2 complex require the presence of a highly acidic 23-residue region at the C terminus of Ubc2. Ubc2-C88A, an inactive variant of Ubc2 in which the active-site Cys-88 has been replaced by Ala, is shown to retain the affinity for N-recognin. Expression of Ubc2-C88A inhibits the N-end rule pathway, apparently as a result of competition between Ubc2 and Ubc2-C88A for binding to N-recognin. The two-hybrid (interaction cloning) technique was used to identify a approximately 170-residue C-terminal fragment of the 1,950-residue N-recognin as a Ubc2-interacting domain. We also show that the level of UBR1 mRNA decreases upon overexpression of UBC2. This effect of UBC2 is observed with cells whose UBR1 is expressed from an unrelated promoter but is not observed if UBR1 contains a frameshift mutation, or if the Ubc2 protein lacks its C-terminal acidic region. The N-recognin.Ubc2 complex appears to regulate the expression of N-recognin through changes in the metabolic stability of its mRNA.
Assuntos
Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases , Alelos , Northern Blotting , Clonagem Molecular , Proteínas Fúngicas/genética , Marcadores Genéticos , Ligases/genética , Plasmídeos , Ligação Proteica , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas Recombinantes/metabolismo , Enzimas de Conjugação de UbiquitinaRESUMO
The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway.