RESUMEN
Endoplasmic reticulum (ER) quality control (ERQC) components retain and degrade misfolded proteins, and our results have found that the degradation of the soluble ERQC substrates CPY* and PrA* but not membrane spanning ERQC substrates requires transport between the ER and Golgi. Stabilization of these misfolded soluble proteins was seen in cells lacking Erv29p, a probable Golgi localized protein that cycles through the ER by means of a di-lysine ER retrieval motif (KKKIY). Cells lacking Erv29p also displayed severely retarded ER exit kinetics for a subset of correctly folded proteins. We suggest that Erv29p is likely involved in cargo loading of a subset of proteins, including soluble misfolded proteins, into vesicles for ER exit. The stabilization of soluble ERQC substrates in both erv29Delta cells and sec mutants blocked in either ER exit (sec12) or vesicle delivery to the Golgi (sec18) suggests that ER-Golgi transport is required for ERQC and reveals a new aspect of the degradative mechanism.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Carboxipeptidasas/metabolismo , Proteínas Portadoras/fisiología , Catepsina A , Eliminación de Gen , Genes Fúngicos , Datos de Secuencia Molecular , Pliegue de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Regulación hacia Arriba , Proteínas de Transporte VesicularRESUMEN
The endoplasmic reticulum quality control (ERQC) system retains and degrades soluble and membrane proteins that misfold or fail to assemble. Vph1p is the 100 kDa membrane subunit of the yeast Saccharomyces cerevisiae V-ATPase, which together with other subunits, assembles into the V-ATPase in the ER, requiring the ER resident protein Vma22p. In vma22Delta cells, Vph1p remains an integral membrane protein with wild-type topology in the ER membrane before undergoing a rapid and concerted degradation requiring neither vacuolar proteases nor transport to the Golgi. Failure to assemble targets Vph1p for degradation in a process involving ubiquitylation, the proteasome and cytosolic but not ER lumenal chaperones. Vph1p appears to possess the traits of a 'classical' ERQC substrate, yet novel characteristics are involved in its degradation: (i) UBC genes other than UBC6 and UBC7 are involved and (ii) components of the ERQC system identified to date (Der1p, Hrd1p/Der3p and Hrd3p) are not required. These data suggest that other ERQC components must exist to effect the degradation of Vph1p, perhaps comprising an alternative pathway.
Asunto(s)
Retículo Endoplásmico/metabolismo , Complejo de la Endopetidasa Proteasomal , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas , ATPasas de Translocación de Protón Vacuolares , Citosol/metabolismo , Metabolismo Energético , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , Control de Calidad , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Ubiquitinas/metabolismoRESUMEN
BACKGROUND: Serum digoxin concentrations (SDCs) are frequently sampled before completion of drug distribution. If elevated, these concentrations may be misinterpreted, potentially leading to a misdiagnosis of digoxin toxicity. OBJECTIVES: To determine the frequency of elevated SDCs (>2.6 nmol/L [>2.0 ng/mL]) obtained at appropriate postdosing intervals and to evaluate the frequency of clinically defined digoxin toxicity in patients with elevated SDCs. METHODS: The medical records of adult patients with SDCs assayed at 5 general hospitals in North Carolina during a 3-month period (May 1 through July 31, 1996) were prospectively evaluated. Data on SDC, inpatient or outpatient status, and medical or surgical service were collected for all patients. Data on patient demographics, serum chemistry values, indication for digoxin treatment, clinical evidence of digoxin toxicity, and timing of the blood sample relative to administration of the last dose of digoxin were collected for patients with SDCs higher than 2.6 nmol/L (>2.0 ng/mL). RESULTS: Of 3434 SDCs assayed in 2009 patients, 320 (9.3%) were higher than 2.6 nmol/L (>2.0 ng/mL). Fifty-one (15.9%) of the 320 SDCs were drawn at 6 hours or less following a digoxin dose. Sampling time relative to the digoxin dose could not be determined in 70 (21.9%) of the 320 elevated SDCs, leaving 199 (62.2%) of 320 SDCs in 138 patients evaluable for digoxin toxicity. Eighty-three of the 138 patients had clinical evidence of digoxin toxicity for an overall incidence of 4.1%. CONCLUSIONS: Digoxin toxicity occurs less frequently than historically reported. Continued emphasis needs to be placed on obtaining appropriately timed SDCs.
Asunto(s)
Digoxina/sangre , Adulto , Diagnóstico Diferencial , Digoxina/uso terapéutico , Femenino , Hospitales Generales , Humanos , Masculino , Persona de Mediana Edad , North Carolina , Intoxicación/diagnóstico , Factores de TiempoRESUMEN
VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.
Asunto(s)
Proteínas Fúngicas/metabolismo , Aparato de Golgi/enzimología , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Vacuolas/enzimología , Proteínas de Transporte Vesicular , Ácido Aspártico Endopeptidasas/metabolismo , Carboxipeptidasas/metabolismo , Catepsina A , Citosol/química , Citosol/enzimología , Proteínas Fúngicas/genética , Aparato de Golgi/química , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fenotipo , Unión Proteica/fisiología , Receptores de Superficie Celular/genética , Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Tirosina/fisiología , Vacuolas/químicaRESUMEN
OBJECTIVE: To describe delayed peak lithium concentrations after an overdose of extended-release lithium tablets. CASE SUMMARY: A patient with borderline personality disorder and depression ingested extended-release lithium approximately 20.25 g along with other agents. At presentation, the lithium concentration was 1.4 mEq/L. Significant enteral intake was initiated 27 hours after presentation and the lithium concentration 5 hours later increased to 3.2 mEq/L. A second lithium peak concentration of 5.0 mEq/L was noted 40 hours after presentation. Two hemodialysis sessions lasting 4 hours each were performed along with administration of sodium polystyrene sulfonate in sorbitol 20% to enhance lithium elimination and decrease absorption. Eighty-eight hours after presentation, the lithium concentration had decreased to 1.5 mEq/L. DISCUSSION: Delayed and secondary peak lithium concentrations have been reported following an overdose with an extended-release product. Extended-release lithium may form an aggregate in the gastrointestinal tract and/or have delayed absorption secondary to coingested drugs. Toxicity may result if the patient begins enteral intake of drugs, fluids, or nutrition. CONCLUSIONS: Continued monitoring of lithium concentrations after an acute ingestion with an extended-release product are recommended until lithium concentrations are less than 1.5 mEq/L and there are no signs of toxicity, particularly once the patient begins significant enteral intake.
Asunto(s)
Antidepresivos/envenenamiento , Litio/envenenamiento , Adulto , Antidepresivos/administración & dosificación , Antidepresivos/sangre , Preparaciones de Acción Retardada , Humanos , Litio/administración & dosificación , Litio/sangre , MasculinoRESUMEN
Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.
Asunto(s)
Endocitosis/fisiología , Proteínas Fúngicas/fisiología , Receptores Acoplados a Proteínas G , Receptores de Feromonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores , Carboxipeptidasas/metabolismo , Catepsina A , Compartimento Celular/fisiología , Clonación Molecular , Proteínas Fúngicas/metabolismo , Expresión Génica/fisiología , Genes Fúngicos/fisiología , Aparato de Golgi/fisiología , Datos de Secuencia Molecular , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Conjugación , Vacuolas/ultraestructuraRESUMEN
Protein splicing is a newly discovered process that is the protein equivalent of RNA splicing. Protein splicing proceeds through a branched protein intermediate, and in vitro studies indicate that the reaction is autocatalytic. The excised 'intein' proteins are site-specific DNA endonucleases that catalyse genetic mobility of their DNA coding sequence by an 'intein homing' mechanism.
Asunto(s)
Proteínas/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Empalme del ARNRESUMEN
Protein splicing is an extraordinary post-translational reaction that removes an intact central "spacer" domain (Sp) from precursor proteins (N-Sp-C) while splicing together the N- and C-domains of the precursor, via a peptide bond, to produce a new protein (N-C). All of the available data on protein splicing fit a model in which these intervening sequences excise at the protein level via a self-splicing mechanism. Several proteins have recently been discovered that undergo protein splicing, and in two such cases, the excised spacer protein is an endonuclease. Such endonucleases are capable of conferring genetic mobility upon the intervening sequences that encodes them. These intervening sequences define a new family of mobile genetic elements that are translated yet remain phenotypically silent by excising at the protein rather than the RNA level.
Asunto(s)
Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ATPasas de Translocación de Protón , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Archaea/genética , Archaea/metabolismo , Concanavalina A/biosíntesis , ADN Polimerasa Dirigida por ADN/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Genes Bacterianos , Genes Fúngicos , Modelos Biológicos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Rec A Recombinasas/biosíntesis , Rec A Recombinasas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.
Asunto(s)
Proteínas Fúngicas/metabolismo , Procesamiento Proteico-Postraduccional , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Precursores de Proteínas/metabolismo , Eliminación de SecuenciaRESUMEN
Over a 5.5 year period, 1264 consecutive patients with acute myocardial infarction as confirmed by enzyme levels were prospectively identified. Of these, 25 (2%) suffered ventricular septal rupture (pulmonary/systemic flow range 1.5 to 6) 7 +/- 7 days after onset of myocardial infarction. Death occurred in 14 patients (56%) and was more common after inferior than anterior myocardial infarction (11 of 15 [73%] vs three of 10 [30%], p less than .05). Among 133 variables analyzed, survivors and nonsurvivors were similar with respect to all premorbid clinical characteristics, infarct size as assessed by peak creatine kinase values, shunt size, two-dimensional echocardiographic and hemodynamic indexes of left ventricular function, and extent of coronary disease. Compared with survivors, the nonsurvivors had greater impairment of right ventricular function as determined by a higher two-dimensional echocardiographically derived right ventricular wall motion index (RVWMI) (0.55 +/- 0.87 vs 1.70 +/- 0.45, p less than .001), greater elevation of right ventricular end-diastolic pressure (11 +/- 6 vs 17 +/- 6, p less than .02), and greater mean right atrial pressure (10 +/- 6 vs 16 +/- 3, p less than .01). Of interest, two of the three patients who presented with anterior myocardial infarction and who died had inferiorly extended infarcts and all had abnormal RVWMIs (greater than or equal to 1.0). As expected, cardiogenic shock shortly after onset of ventricular septal rupture was associated with a 91% mortality, but was more common after inferior than anterior myocardial infarction (60% vs 20%, p less than .05). The mean effective cardiac index was also higher in survivors than nonsurvivors (2.1 +/- 0.5 vs 1.2 +/- 0.5, p less than .001). Finally, multivariate analysis indicated that all nonsurvivors could be identified based on: an effective cardiac index of 1.75 liters/min/m2 or less, the presence of extensive right ventricular and septal dysfunction on the two-dimensional echocardiogram, a mean right atrial pressure of 12 mm Hg or more, and early onset of ventricular septal rupture. Thus, our data demonstrate that: mortality is higher when ventricular septal rupture complicates inferior than when it complicates anterior myocardial infarction, survivors can be distinguished from nonsurvivors and the prediction of outcome is highly accurate, and combined right ventricular and septal dysfunction has a substantial impact on prognosis.
Asunto(s)
Rotura Cardíaca/etiología , Tabiques Cardíacos , Corazón/fisiopatología , Infarto del Miocardio/complicaciones , Adulto , Anciano , Análisis de Varianza , Angiografía , Presión Sanguínea , Gasto Cardíaco , Ecocardiografía , Femenino , Estudios de Seguimiento , Rotura Cardíaca/mortalidad , Rotura Cardíaca/fisiopatología , Hemodinámica , Humanos , Masculino , Persona de Mediana Edad , Contracción Miocárdica , Infarto del Miocardio/mortalidad , Infarto del Miocardio/fisiopatología , Pronóstico , Estudios Prospectivos , Choque Cardiogénico/mortalidad , Volumen SistólicoRESUMEN
To elucidate the functional and prognostic significance of right ventricular dysfunction after acute inferior wall myocardial infarction, 74 consecutive patients with inferior infarction were prospectively evaluated with gated equilibrium blood pool imaging at rest, submaximal exercise thallium-201 scintigraphy and coronary angiography before hospital discharge. In addition, symptom-limited stress thallium-201 scintigraphy was performed in 61 patients at 3 months, and all patients were followed up clinically for 23 +/- 15 months. Utilizing predetermined radionuclide angiographic criteria, 47 patients (Group I) had normal right ventricular function, 12 patients (Group II) had mild to moderate dysfunction and 15 patients (Group III) had severe right ventricular dysfunction. There were no significant differences among the groups with regard to age, history of prior myocardial infarction, peak creatine kinase values, maximal Killip functional class, number or type of in-hospital complications, left ventricular ejection fraction, prevalence of multivessel disease or the distribution and severity of disease affecting the infarct-related vessel. Exercise tolerance as assessed by treadmill time, blood pressure-heart rate product and peak work load in METS was comparable among the three groups, both before hospital discharge and at 3 month follow-up. No differences in indicators of exercise-induced ischemia were noted among the groups, including the prevalence of redistribution thallium-201 defects, ST segment depression or symptoms of chest pain. Finally, cardiac mortality, reinfarction rate and the incidence of medically refractory angina pectoris were similar in the three groups. Thus, right ventricular dysfunction after acute inferior wall myocardial infarction does not appear to limit exercise tolerance or identify a subgroup of patients at higher risk for recurrent cardiac events.