scholarly journals Role of BGS13 in the Secretory Mechanism of Pichia pastoris

2019 ◽  
Vol 85 (24) ◽  
Author(s):  
Christopher A. Naranjo ◽  
Anita D. Jivan ◽  
Maria N. Vo ◽  
Katherine H. de Sa Campos ◽  
Jared S. Deyarmin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pichia Pastoris ◽  
Mutant Strain ◽  
Recombinant Proteins ◽  
Economic Benefits ◽  
Heterologous Protein Expression ◽  
Content Type ◽  
Kinase C

ABSTRACT The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

scholarly journals Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Asumi Sugahara ◽  
Akira Yoshimi ◽  
Fumio Shoji ◽  
Tomonori Fujioka ◽  
Kiyoshi Kawai ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Filamentous Fungi ◽  
Magnaporthe Grisea ◽  
Rice Blast Fungus ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Kinase C ◽  
Mitogen Activated Protein

ABSTRACTThe cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungusAspergillus nidulansand in MgPkc1p in the rice blast fungusMagnaporthe griseaare lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried outin silicoscreening by three-dimensional (3D) structural modeling and performed growth inhibition tests forM. griseaon agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed inSaccharomyces cerevisiaecells in which the kinase domain of nativeS. cerevisiaePKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis ofMLP1, which encodes a downstream factor of PKC inS. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress inM. grisea. We discuss the relationships between PKC and melanin biosynthesis.IMPORTANCEA candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified byin silicoscreening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed inSaccharomyces cerevisiae. Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison withS. cerevisiaePKC. Analysis of theAGS1mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungusMagnaporthe grisearevealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization inM. grisea, and this melanization, which is required for pathogenicity ofM. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of4HNR,3HNR, andSCD1, which are essential for melanization inM. grisea, were suppressed by both PKC inhibitors.

scholarly journals Direct evidence for a key role of protein kinase C in the development of vasospasm after subarachnoid hemorrhage

1992 ◽  
Vol 76 (4) ◽  
pp. 635-639 ◽  
Author(s):  
Shigeru Nishizawa ◽  
Nobukazu Nezu ◽  
Kenichi Uemura
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Evidence ◽  
Control Group ◽  
Content Type ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Fine Print

✓ Vascular contraction is induced by the activation of intracellular contractile proteins mediated through signal transduction from the outside to the inside of cells. Protein kinase C plays a crucial role in this signal transduction. It is hypothesized that protein kinase C plays a causative part in the development of vasospasm after subarachnoid hemorrhage (SAH). To verify this directly, the authors measured protein kinase C activity in canine basilar arteries in an SAH model with (γ-32P)adenosine triphosphate and the data were compared to those in a control group. Protein kinase C is translocated to the membrane from the cytosol when it is activated, and the translocation is an index of the activation; thus, protein kinase C activity was measured both in the cytosol and in the membrane fractions. Protein kinase C activity in the membrane in the SAH model was remarkably enhanced compared to that in the control group. The percentage of membrane activity to the total was also significantly greater in the SAH vessels than in the control group, and the percentage of cytosol activity in the SAH group was decreased compared to that in the control arteries. The results indicate that protein kinase C in the vascular smooth muscle was translocated to the membrane from the cytosol and was activated when SAH occurred. It is concluded that this is direct evidence for a key role of protein kinase C in the development of vasospasm.

Response of malignant glioma cell lines to activation and inhibition of protein kinase C-mediated pathways

1990 ◽  
Vol 73 (1) ◽  
pp. 98-105 ◽  
Author(s):  
Ian F. Pollack ◽  
Margaret S. Randall ◽  
Matthew P. Kristofik ◽  
Robert H. Kelly ◽  
Robert G. Selker ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Malignant Glioma ◽  
Polymyxin B ◽  
Content Type ◽  
Rat Glioma ◽  
Kinase C ◽  
Low Passage

✓ To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and C6), and two low-passage human glioma lines (obtained from surgical specimens) were studied. With the exception of the C6 line, all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma. At optimal concentrations (5 to 10 ng/ml), TPA produced a two- to five-fold increase in the rate of DNA synthesis (p < 0.05) as assessed by incorporation of 3H-thymidine. However, TPA had no additive effect on the mitogenic response produced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). Inhibition of PKC using the antibiotic polymyxin B (20 µg/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.

scholarly journals The Yeast Protein Kinase C Cell Integrity Pathway Mediates Tolerance to the Antifungal Drug Caspofungin through Activation of Slt2p Mitogen-Activated Protein Kinase Signaling

2003 ◽  
Vol 2 (6) ◽  
pp. 1200-1210 ◽  
Author(s):  
Cristina Reinoso-Martín ◽  
Christoph Schüller ◽  
Manuela Schuetzer-Muehlbauer ◽  
Karl Kuchler
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Antifungal Drug ◽  
Mitogen Activated Protein Kinase ◽  
Cell Integrity ◽  
Cell Wall Synthesis ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Yeast Genes

ABSTRACT The echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of β-(1-3)-glucan synthesis. Saccharomyces cerevisiae cells are able to tolerate rather high caspofungin concentrations, displaying high viability at low caspofungin doses. To identify yeast genes implicated in caspofungin tolerance, we performed a genome-wide microarray analysis. Strikingly, caspofungin treatment rapidly induces a set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required for cell wall maintenance and architecture. The mitogen-activated protein kinase Slt2p is rapidly activated by phosphorylation, triggering signaling through the PKC pathway. Cells lacking genes such as SLT2, BCK1, and PKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating that the PKC pathway is required for caspofungin tolerance. Notably, the cell surface integrity sensor Wsc1p, but not the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations. The expression modulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of several cell wall synthesis and maintenance genes. Thus, caspofungin-induced cell wall damage requires Wsc1p as a dedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wall synthesis. Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker's yeast.

Generation of the catalytic fragment of protein kinase C alpha in spastic canine basilar artery

1997 ◽  
Vol 87 (5) ◽  
pp. 752-756 ◽  
Author(s):  
Motohiko Sato ◽  
Eiichi Tani ◽  
Tsuyoshi Matsumoto ◽  
Hirokazu Fujikawa ◽  
Shinobu Imajoh-Ohmi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Basilar Artery ◽  
Cleavage Site ◽  
Limited Proteolysis ◽  
Specific Inhibitor ◽  
Content Type ◽  
Kinase C ◽  
Canine Basilar Artery ◽  
Catalytic Fragment

✓ In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated µ-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCα antibody shows a significant decrease in cytosolic 80-kD PKCα and a concomitantly significant increase in membrane PKCα in the spastic canine BA. In addition, an immunoblot study in which cleavage site—directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKCα and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site—directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKCα in spastic canine BA is translocated to the cell membrane, where PKCα is rapidly cleaved into PKM as a result of proteolysis of the isozyme by µ-calpain but not by µ-calpain. The authors hypothesize that µ-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCα.

scholarly journals The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

1996 ◽  
Vol 16 (12) ◽  
pp. 6752-6764 ◽  
Author(s):  
T Toda ◽  
S Dhut ◽  
G Superti-Furga ◽  
Y Gotoh ◽  
E Nishida ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Amino Acid ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Mitogen Activated Protein Kinase ◽  
Cell Integrity ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Mapk Gene

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.

Protein kinase C and diacylglycerol content in basilar arteries during experimental cerebral vasospasm in the dog

1995 ◽  
Vol 82 (5) ◽  
pp. 834-840 ◽  
Author(s):  
Masayuki Yokota ◽  
John W. Peterson ◽  
Marios C. Kaoutzanis ◽  
Kenta Yamakawa ◽  
Robert Sibilia ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cerebral Vasospasm ◽  
Blood Clot ◽  
Statistical Significance ◽  
Content Type ◽  
Control Value ◽  
Kinase C ◽  
Basilar Arteries ◽  
Subarachnoid Blood

✓ Sixteen dogs were entered into a study of the double subarachnoid hemorrhage (SAH) model of cerebral vasospasm. Six animals were sacrificed 72 hours after the first experimental SAH, and the remaining 10 animals were killed 72 hours after the second experimental SAH; ten additional animals served as controls. Basilar arteries were rapidly excised from the dogs and frozen. Multiple segments of the frozen arteries were analyzed independently for total protein and 1,2-diacylglycerol (DAG) content, which averaged 3.17 (± 0.27 standard error of the mean; SEM) pmol DAG/µg protein for all 25 arteries analyzed. A slight decreasing trend in DAG content relative to that of control vessels was found in vessels chronically constricted in situ by subarachnoid blood clot; however, this trend did not attain statistical significance. Two segments of the same vessels were assayed independently for protein kinase C (PKC) activity, which averaged 1.21 (± 0.08 SEM) pmol phosphate incorporation per minute per µg protein for all 24 arteries analyzed. A small decrease in PKC content was noted in vessels that experienced a single SAH; however, PKC returned to near control value in vessels subjected to double SAH. The ratio of particulate (membrane bound) to soluble PKC activity, an indicator of PKC translocation to the membrane and hence PKC activation, showed a small but statistically significant trend to increase with experimental SAH.

scholarly journals PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

PLoS Genetics ◽  
2014 ◽  
Vol 10 (9) ◽  
pp. e1004582 ◽  
Author(s):  
Ines Teichert ◽  
Eva Katharina Steffens ◽  
Nicole Schnaß ◽  
Benjamin Fränzel ◽  
Christoph Krisp ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Scaffold Protein ◽  
Cell Wall Integrity ◽  
Activator Protein ◽  
Kinase C ◽  
Cell Wall Integrity Pathway

scholarly journals Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

2017 ◽  
Vol 85 (7) ◽  
Author(s):  
Manmeet Bhalla ◽  
Daria Law ◽  
Georgina C. Dowd ◽  
Keith Ireton
Keyword(s):  
Protein Kinase C ◽  
Listeria Monocytogenes ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Bacterial Pathogen ◽  
Human Cell Line ◽  
Content Type ◽  
Kinase C ◽  
Cell Line Hela ◽  
Threonine Kinases

ABSTRACT The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-α (PKC-α) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1α (HIF-1α) and the mTORC2 substrate PKC-α each control Listeria uptake. Genetic or pharmacological inhibition of PKC-α reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-α to be host factors exploited by Listeria to promote infection. PKC-α controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.

The effects of papaverine on phorbol dibutyrate-induced vasoconstriction in brain slice microvessels

1994 ◽  
Vol 81 (4) ◽  
pp. 574-578 ◽  
Author(s):  
Yongcheng Jin ◽  
Oren Sagher ◽  
Quoc-Anh Thai ◽  
Neal F. Kassell ◽  
Kevin S. Lee
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cerebral Vasospasm ◽  
Cerebral Microvessels ◽  
Content Type ◽  
Phorbol Dibutyrate ◽  
Kinase C ◽  
Significant Reversal ◽  
Dose Dependent

✓ Papaverine (PPV) is a nonspecific vasodilator with widespread clinical uses in the treatment of arterial spasm. It has also been utilized in an attempt to reverse cerebral vasospasm. Recent angiographic results have demonstrated significant reversal of vasospasm in large vessels after selective intra-arterial application of PPV; however, these impressive results lacked good clinical correlation. In this study, phorbol dibutyrate was used to stimulate protein kinase C in an in vitro model of cerebral microvessels. Papaverine was found to elicit a dose-dependent exacerbation of phorbol dibutyrate-induced microvascular constriction in this model system. Because protein kinase C is thought to play a key role in the development of cerebral vasospasm, PPV-induced vasoconstriction represents a potentially important deleterious effect that may not be apparent on angiography. Such a constrictor response may compromise the beneficial vasodilatory effect seen with intra-arterial injection of PPV.

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