RESUMEN
We studied the efficacy and safety of the combined treatment with programmed cell death 1 (PD-1) inhibitors and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy and subsequent PD-1 inhibitor maintenance treatment in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and high tumor burden. Forty-four R/R DLBCL patients with high tumor burden were enrolled in this study. The experimental group of 26 patients received combined therapy with PD-1 inhibitors and anti-CD19-CAR T cells, while the control group of 18 patients received anti-CD19-CAR T-cell therapy alone. The objective response rate (ORR) was 65.39% and 61.11% in the combination and control groups, respectively. The PD-1 inhibitor maintenance therapy was selected for patients who achieved complete response or partial response in the combination therapy group. Progression-free survival and overall survival rates in the combination group were higher than those in the control group 3 and 12 months after CAR T-cell infusion. There was no significant difference in the grade of cytokine release syndrome or immune effector cell associated neurotoxic syndrome between the two groups. In the maintenance therapy group, only eight patients experienced grade 1 Common Terminology Criteria for Adverse Events (CTCAE) and three grade 2 CTCAE. Overall, we found that the ORR was not affected by the combination therapy with PD-1 inhibitors and anti-CD19-CAR T cells. However, patients who had achieved the ORR might benefit from PD-1 inhibitor maintenance therapy after combination therapy without increased side effects.
Asunto(s)
Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Carga Tumoral , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Antígenos CD19 , Linfocitos T , ApoptosisRESUMEN
A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.
Asunto(s)
Benzofuranos/química , Benzofuranos/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Benzofuranos/administración & dosificación , Benzofuranos/farmacocinética , Benzofuranos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
This Letter describes the discovery of a series of potent inhibitors of Human Uric Acid Transporter 1 (hURAT1). Lead generation and optimization via 3D pharmacophore analysis resulted in compound 41. With an IC50 of 33.7nM, 41 also demonstrated good oral bioavailability in rat (74.8%) and displayed a consistent PK profile across all species tested (rat, dog and monkey).
Asunto(s)
Ciclobutanos/farmacología , Supresores de la Gota/farmacología , Transportadores de Anión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Ciclobutanos/síntesis química , Ciclobutanos/farmacocinética , Perros , Supresores de la Gota/síntesis química , Supresores de la Gota/farmacocinética , Humanos , Macaca fascicularis , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Transportadores de Anión Orgánico/química , Proteínas de Transporte de Catión Orgánico/química , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
RESUMEN
During membrane biogenesis, the M13 procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner with both the hydrophobic signal sequence and the membrane anchor sequence promoting translocation of the periplasmic loop via a hairpin mechanism. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. When the polarity of the translocated peptide region is lowered and the charged residues in this region are removed, translocation of this loop region occurs largely by a YidC- and Sec-independent mechanism. When the polarity is increased to that of the wild-type procoat protein, the YidC insertase is essential for translocation. Further increasing the polarity, by adding charged residues, switches the insertion pathway to a YidC/Sec mechanism. Conversely, we find that increasing the hydrophobicity of the transmembrane segments of PClep can decrease the translocase requirement for translocation of the peptide chain. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own.
Asunto(s)
Proteínas de la Cápside/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Membrana Celular/virología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutación , Periplasma/química , Periplasma/metabolismo , Canales de Translocación SEC , Proteína SecARESUMEN
The MAPK pathway is identified as one of the most important pathways involved in cell proliferation and differentiation. A key kinase in the pathway, the Mitogen-activated protein kinase kinase (MEK) is recognized as a promising target for antitumor drugs. Structure-based design and optimization of known MEK inhibitors resulted in identification of compound 10a as a potent non-ATP competitive MEK inhibitor in both in vitro and in vivo tests.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Diseño de Fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Experimentales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Células HCT116 , Humanos , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this Letter we describe SAR investigation on the cyclopentyl-triazolol-pyrimidine scaffold in pursuit of new oral P2Y12 inhibitors. Different synthetic routes were developed for variations at the cyclopentyl core. Optimization finally led to compound 2d which was advanced into preclinical development based on better potency and safety profile in comparison to ticagrelor.
Asunto(s)
Ciclopentanos/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Pirimidinas/farmacología , Receptores Purinérgicos P2Y12/metabolismo , Triazoles/farmacología , Administración Oral , Animales , Ciclopentanos/administración & dosificación , Ciclopentanos/química , Perros , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Agregación Plaquetaria/efectos de los fármacos , Plasma Rico en Plaquetas/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/administración & dosificación , Antagonistas del Receptor Purinérgico P2Y/química , Pirimidinas/administración & dosificación , Pirimidinas/química , Ratas , Relación Estructura-Actividad , Triazoles/administración & dosificación , Triazoles/químicaRESUMEN
The GPR40 (FFA1) has emerged as an attractive target for a novel insulin secretagogue with glucose dependency. A series of novel orally bioavailable GPR40 agonists was discovered. SAR study and structural optimization led to identification of compounds 28a and 30a as potent GPR40 agonists with superior physiochemical properties and robust in vivo efficacy in rhesus monkeys.
Asunto(s)
Benzofuranos/farmacología , Descubrimiento de Drogas , Receptores Acoplados a Proteínas G/agonistas , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/farmacología , Administración Oral , Animales , Benzofuranos/administración & dosificación , Benzofuranos/química , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Humanos , Macaca mulatta , Ratones , Modelos Moleculares , Estructura Molecular , Ratas , Receptores Acoplados a Proteínas G/genética , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Sulfonas/administración & dosificación , Sulfonas/químicaRESUMEN
In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-negative bacteria, and Gram-positive bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the number of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Archaea/enzimología , Proteínas Arqueales/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/química , Proteínas Arqueales/química , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecARESUMEN
Post-transplant lymphoproliferative disease (PTLD) often exhibits poor prognosis and high mortality, and there are no uniform guidelines for the treatment of this disease. Anti-CD19 chimeric antigen receptor (CAR) T cells show significant efficacy in treatment of relapse/refractory diffuse large B-cell lymphoma (DLBCL). Treatment using anti-CD19-CAR T-cell therapy in PTLD has been limited by immunosuppressants and has not been widely employed. In this study, a refractory post kidney transplant DLBCL patient with a high tumor burden was enrolled in a clinical trial of anti-CD19-CAR T-cell therapy. The tacrolimus dose was not decreased during combination chemotherapy, as the creatinine level of the patient increased. To improve the function of autologous T cells, combination therapy with anti-CD19-CAR T cells and programmed cell death 1 (PD-1) inhibitors was selected. After treatment with the combination therapy, the patient was diagnosed with grade 1 cytokine release syndrome and grade 3 immune effector cell-associated neurotoxicity syndrome. The amplification peak of anti-CD19-CAR T cells reached 9.01% on day 7. With PD-1 inhibitor maintenance therapy, his disease was maintained in partial remission for 18 weeks. However, his tumor suddenly increased in size, and he discontinued the treatment, including radiation therapy. The anti-CD19-CAR T cell and PD-1 inhibitors have a combined effect on PTLD, and this combination therapy needs to be further explored.
RESUMEN
In recent years, many new treatments for relapsed/refractory (R/R) multiple myeloma (MM) have improved patient prognosis, but the prognosis of patients with extramedullary MM is still particularly poor. Therefore, more efficacious therapies and novel strategies are urgently needed for these patients. The aim of this study was to observe and compare the efficacy and safety of humanized anti-B cell maturation antigen (anti-BCMA) chimeric antigen receptor (CAR) T cell therapy in R/R MM patients with and without extramedullary disease. Seven R/R MM patients with extramedullary disease and 13 without extramedullary disease received humanized anti-BCMA CAR T cell therapy. The overall response rate was not different between patients with and without extramedullary disease. There was no difference in the progression-free survival (PFS) or overall survival (OS) rates between the two groups at 180 days, but the PFS and OS rates in patients with extramedullary disease were lower at 360 days than those in patients without extramedullary disease. Although some patients with extramedullary disease experienced further disease progression, their M protein level did not increase. We did not see this change trend of M protein in patients without extramedullary disease. However, this was not observed in patients without extramedullary disease. Among patients who responded to CAR T cell therapy, the grades of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxic syndrome (ICANS) were much higher among patients with extramedullary disease. In summary, R/R MM patients with extramedullary disease could benefit from humanized anti-BCMA CAR T cell therapy in the short term, although the CRS and ICANS grades were much higher in patients with extramedullary disease. Therefore, anti-BCMA CAR T cell therapy allows for a remission time for R/R MM patients with extramedullary disease, which could be maintained by bridging hematopoietic stem cell transplantation, radiotherapy, and other therapies. Clinical Trial Registration: http://www.chictr.org.cn/index.aspx, identifiers ChiCTR1800017051 and ChiCTR2000033925.
Asunto(s)
Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/inmunología , Adulto , Anciano , Antígeno de Maduración de Linfocitos B/inmunología , Comorbilidad , Manejo de la Enfermedad , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunoterapia Adoptiva/efectos adversos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/mortalidad , Metástasis de la Neoplasia , Pronóstico , Receptores Quiméricos de Antígenos/genética , Recurrencia , Retratamiento , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
PURPOSE: To investigate the donor chimerism changes and curative effects associated with the use of autologous anti-CD19 chimeric antigen receptor (CAR) T cells with B-cell acute lymphoblastic leukemia (B-ALL) presenting with a low donor chimerism level and relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT). METHODS: Nine patients with B-ALL showing low donor chimerism level and relapse after allo-HSCT were enrolled. Patients 1-3 received CD19 CAR-T cell therapy using cells derived from autologous peripheral blood mononuclear cells (PBMCs) (comprising a mixture of patient and original donor cells) as their donors could not provide PBMCs. Samples from the other six patients (Patients A-F) were investigated only in vitro. The changes in the degree of donor chimerism, function of the CD19 CAR-T cells and T cells in all nine patients were analyzed in vitro. The therapeutic effects and adverse events (AEs) were also evaluated in Patients 1-3. RESULTS: The CAR-T cells and T cells in all nine patients showed complete donor chimerism restoration following a 12-day culture period in vitro. These CD19 CAR-T cells demonstrated strong cytotoxicity towards Nalm 6 cells in vitro except in patients 3 and D. In the latter patients, the absolute numbers of all subsets, especially the CD8 + T-cell absolute numbers in peripheral blood were very low. Patients 3 and D showed relatively short durations from transplant to recurrence and received chemotherapy after relapse. In the patients receiving CD19 CAR-T cell therapy, the most commonly observed AE was grade 1 to 2 cytokine release syndrome. None of the cases showed acute graft-versus-host disease during treatment. Patients 1 and 2 achieved complete response with complete restoration of donor chimerism. Patient 3, who received the same CD19 CAR-T cell therapy, did not respond to this therapy. CONCLUSION: CD19 CAR-T cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for salvage therapy and could restore to complete donor chimerism after 12 days' culture in vitro. TRIAL REGISTRATION: Humanized CD19 CAR-T cell therapy for relapse or refractory B-cell lymphoma or acute B lymphocytic leukemia, ChiCTR1800019622, Registered 24 November 2018, http://www.chictr.org.cn/index.aspx.
RESUMEN
The coat protein of bacteriophage Pf3 is inserted into the plasma membrane of Escherichia coli by the insertase YidC. To identify which of the six transmembrane regions of YidC bind the single-spanning Pf3 coat protein during membrane protein biogenesis, we used the disulfide cross-linking approach. We generated single cysteines in each of the transmembrane regions of YidC and in the center of the hydrophobic region of Pf3 coat protein. We found that the substrate Pf3 coat contacts the first and third transmembrane segment (TM) of YidC as crosslinks between these two proteins can be formed in vivo during membrane biogenesis. A detailed disulfide-mapping study revealed that one face of TM3 of YidC makes contact with the Pf3 protein.
Asunto(s)
Proteínas de la Cápside/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Membrana Celular/ultraestructura , Reactivos de Enlaces Cruzados , Cisteína/química , Cisteína/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Mutación , Mapeo de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the yidC(C423R) mutant confers a weak phenotype on membrane protein insertion while a yidC(P431L) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F(1)F(o) subunit c (F(o)C), while CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that -3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while F(o)C required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación Missense , Sustitución de Aminoácidos , Frío , Análisis Mutacional de ADN , Escherichia coli/crecimiento & desarrollo , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , Supresión GenéticaRESUMEN
Integral membrane proteins typically span the lipid bilayer with hydrophobic alpha helices. These proteins can span the membrane once or multiple times with hydrophilic domains facing both sides of the membrane. In Escherichia coli, the insertion of proteins into the membrane is catalyzed by the Sec translocase and the YidC membrane insertase. YidC can function on its own to insert proteins or together with the Sec translocase to facilitate membrane protein insertion. In this chapter, we will describe the construction of a YidC depletion strain that can be used to examine whether YidC is required for membrane protein insertion. We will also present assays for determining whether a region of a membrane protein is inserted across the membrane.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Inmunoprecipitación , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Transporte de Proteínas/genética , Transporte de Proteínas/fisiologíaRESUMEN
Subunit II (CyoA) of cytochrome bo(3) oxidase, which spans the inner membrane twice in bacteria, has several unusual features in membrane biogenesis. It is synthesized with an amino-terminal cleavable signal peptide. In addition, distinct pathways are used to insert the two ends of the protein. The amino-terminal domain is inserted by the YidC pathway whereas the large carboxyl-terminal domain is translocated by the SecYEG pathway. Insertion of the protein is also proton motive force (pmf)-independent. Here we examined the topogenic sequence requirements and mechanism of insertion of CyoA in bacteria. We find that both the signal peptide and the first membrane-spanning region are required for insertion of the amino-terminal periplasmic loop. The pmf-independence of insertion of the first periplasmic loop is due to the loop's neutral net charge. We observe also that the introduction of negatively charged residues into the periplasmic loop makes insertion pmf dependent, whereas the addition of positively charged residues prevents insertion unless the pmf is abolished. Insertion of the carboxyl-terminal domain in the full-length CyoA occurs by a sequential mechanism even when the CyoA amino and carboxyl-terminal domains are swapped with other domains. However, when a long spacer peptide is added to increase the distance between the amino-terminal and carboxyl-terminal domains, insertion no longer occurs by a sequential mechanism.
Asunto(s)
Complejo IV de Transporte de Electrones/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Insercional , Subunidades de Proteína , Catálisis , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Periplasma/química , Plásmidos , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/química , Subunidades de Proteína/genéticaRESUMEN
The M13 phage Procoat protein is one of the best characterized substrates for the novel YidC pathway. It inserts into the membrane independent of the SecYEG complex but requires the 60 kDa YidC protein. Mutant Procoat proteins with alterations in the periplasmic region had been found to require SecYEG and YidC. In this report, we show that the membrane insertion of these mutants also strongly depends on SecDF that bridges SecYEG to YidC. In a cold-sensitive mutant of YidC, the Sec-dependent function of YidC is strongly impaired. We find that specifically the SecDF-dependent mutants are inhibited in the cold-sensitive YidC strain. Finally, we find that subtle changes in the periplasmic loop such as the number and location of negatively charged residues and the length of the periplasmic loop can make the Procoat strictly Sec-dependent. In addition, we successfully converted Sec-independent Pf3 coat into a Sec-dependent protein by changing the location of a negatively charged residue in the periplasmic tail. Protease mapping of Pf3 coat shows that the insertion-arrested proteins that accumulate in the YidC- or in the SecDF-deficient strains are not translocated. Taken together, the data suggest that the Sec-dependent mutants insert at the interface of YidC and the translocon with SecDF assisting in the translocation step in vivo.