RESUMO
Chronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways is a hallmark of a variety of B-cell lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive JAK/STAT signaling is crucial for survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of cHL. Although the molecular basis of this constitutive JAK/STAT signaling in cHL has not been completely understood, accumulating reports highlight the role of an inactivation or reduced expression of negative JAK/STAT regulators such as silencer of cell signaling 1 (SOCS1) or protein-tyrosine phosphatase 1B (PTP1B) in this process. Here, we report the expression of truncated PTP1B mRNA variants identified in cHL cell lines and primary cHL tumor samples lacking either 1 or several exon sequences. One of these novel PTP1B variants, a splice variant lacking exon 6 (PTP1BΔ6), was found expressed at low levels in cHL cell lines. However, serum stimulation of cHL augmented the expression of PTP1BΔ6 significantly. Functional characterization of PTP1BΔ6 revealed a positive effect on interferon-γ- and interleukin-4-induced JAK/STAT activity in HEK293 or HEK293-STAT6 cells, and on the basal STAT activity in stably transfected L-428 and U-HO1 cHL cell lines. Furthermore, PTP1BΔ6 expression increased the proliferation of L-428 and U-HO1 cells and reduced cytotoxic effects of the chemotherapeutical agents gemcitabine and etoposide distinctively. Collectively, these data indicate that PTP1BΔ6 is a positive regulator of JAK/STAT signaling in cHL.
Assuntos
Doença de Hodgkin/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Transdução de Sinais , Antineoplásicos/farmacologia , Morte Celular , Proliferação de Células , Células HEK293 , Doença de Hodgkin/genética , Humanos , Interferon gama/farmacologia , Interleucina-4/farmacologia , Janus Quinases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/genética , Fatores de Transcrição STAT/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter. RESULTS: The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions. CONCLUSIONS: The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.
Assuntos
Bacillus subtilis/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Fosfatase Alcalina/metabolismo , Bacillus subtilis/química , Escherichia coli/enzimologia , Expressão Gênica , Biblioteca de Peptídeos , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genéticaRESUMO
It is well documented that female minority founders (FMFs) face disadvantages in starting and scaling their ventures. However, the causes of these disadvantages-as well as how FMFs navigate these challenges-are less understood. Our article adopts an intersectionality lens, which allows us to focus on and examine the multiple intersecting dimensions of FMFs (such as gender, ethnicity, migrant status, and social class) and how they influence their entrepreneurial experiences. Drawing upon an inductive study of FMFs operating in Berlin, we build a theory on intersectional identity and image work in startup contexts. We found key structural barriers that serve to sustain inequality and continue to favor more prototypical founders. However, we also identified sources of penalties and privileges that exacerbate (or mitigate) inequality and result in founder image discrepancy. Our analysis demonstrates how founder image discrepancy can prompt FMFs to engage in a progression of intersectional identity and image work that shapes their founder identity. These findings advance entrepreneurship and identity research by extending our understanding of how intersectional identity challenges and opportunities manifest and can be managed within startup contexts. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
RESUMO
Stress-induced degradation of the Bacillus subtilis anti-sigma factor RsiW results in the induction of genes controlled by the extracytoplasmic function sigma factor sigma(W). RsiW is cleaved by the mechanism of regulated intramembrane proteolysis at site-1 and -2 by PrsW and RasP respectively, and is then further degraded by cytoplasmic Clp peptidases. In a reconstituted Escherichia coli system, PrsW removes 40 amino acids from RsiW by cleaving between Ala168 and Ser169 of the extracytoplasmic domain, thereby generating RsiW-S1. Further trimming of RsiW-S1's C-terminus by the periplasmic tail-specific protease Tsp is crucial for subsequent RasP-catalysed clipping. In B. subtilis, mutation of RsiW at Ala168 severely impairs site-1 processing. RsiW-S1 is undetectable in wild-type B. subtilis and knockout strains lacking various extracytoplasmic proteases. While it can be stabilized by C-terminal tagging, even this fusion protein is still attacked. Thus, several peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Overall, the RsiW degradation pathway can be subdivided into two modules each consisting of a site-specific peptidase that prepares RsiW for further degradation by downstream proteases.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação de Sentido IncorretoRESUMO
There is growing evidence that proteolytic degradation of membrane-spanning regulatory proteins such as anti-sigma factors is involved in a variety of important transmembrane signaling processes in bacteria. This mechanism of regulated intramembrane proteolysis (RIP) enables them to respond to extracellular signals and stresses. Here, we summarize current knowledge of RIP controlling extracytoplasmic function sigma factors.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Fator sigma/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Estresse FisiológicoRESUMO
The Bacillus subtilis sigma(W) regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of sigma(W) and the transcription of sigma(W)-controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in sigma(W) induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing alpha-amylase, in competence development, and in formation of multicellular structures known as biofilms.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Peptídeo Hidrolases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Peptídeo Hidrolases/genética , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Genes of Bacillus subtilis controlled by the alternative extracytoplasmic function family sigma factor sigmaW constitute an antibiosis regulon. Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates sigmaW. Upon a stress signal, RsiW is degraded by a mechanism of regulated intramembrane proteolysis. To identify genes which influence RsiW degradation, a transposon screen with a reporter fusion of the green fluorescent protein to RsiW was performed. Among several gene loci identified, the ypdC (prsW) gene displayed a strong effect on RsiW stability. In a ypdC null mutant, induction of sigmaW-controlled genes is abolished and site-1 proteolysis of RsiW is completely blocked. Transcriptional analysis revealed that ypdC is a monocistronic gene, and the defect of sigmaW induction of the null mutant was complemented by ectopically integrated ypdC under xylose control. Orthologues of YpdC can be found in a variety of different bacteria. Its membrane topology was analysed by alkaline phosphatase fusions, revealing that YpdC contains five transmembrane segments and two larger extracytoplasmic loops. In the first loop, two invariantly conserved glutamate residues can be found. In an Escherichia coli system, the cloned ypdC is the only determinant of efficient degradation of RsiW; however, YpdC does not display plain similarities to known proteases, suggesting that it either controls the activity of site-1 proteolysis of RsiW or represents a new type of protease.