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1.
Microb Cell Fact ; 23(1): 99, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38566096

RESUMO

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.


Assuntos
6-Fitase , Saccharomycetales , Pichia/metabolismo , Metanol/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo
2.
Microb Cell Fact ; 22(1): 63, 2023 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-37013612

RESUMO

BACKGROUND: Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast, in search of suitable novel promoters able to drive transcription in response to the pH signal. RESULTS: In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. CONCLUSIONS: This work defines in K. phaffii a subset of genes and diverse cellular pathways that are altered in response to moderate alkalinization of the medium, thus setting the basis for developing novel pH-controlled systems for heterologous protein expression in this fungus.


Assuntos
Ascomicetos , Saccharomycetales , Transcriptoma , Saccharomycetales/genética , Perfilação da Expressão Gênica , Ascomicetos/genética
3.
Int J Mol Sci ; 24(6)2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36982620

RESUMO

The Saccharomyces cerevisiae ENA1 gene, encoding a Na+-ATPase, responds transcriptionally to the alkalinization of the medium by means of a network of signals that involves the Rim101, the Snf1 and PKA kinases, and the calcineurin/Crz1 pathways. We show here that the ENA1 promoter also contains a consensus sequence, located at nt -553/-544, for the Stp1/2 transcription factors, the downstream components of the amino acid sensing SPS pathway. Mutation of this sequence or deletion of either STP1 or STP2 decreases the activity of a reporter containing this region in response to alkalinization as well as to changes in the amino acid composition in the medium. Expression driven from the entire ENA1 promoter was affected with similar potency by the deletion of PTR3, SSY5, or simultaneous deletion of STP1 and STP2 when cells were exposed to alkaline pH or moderate salt stress. However, it was not altered by the deletion of SSY1, encoding the amino acid sensor. In fact, functional mapping of the ENA1 promoter reveals a region spanning from nt -742 to -577 that enhances transcription, specifically in the absence of Ssy1. We also found that the basal and alkaline pH-induced expression from the HXT2, TRX2, and, particularly, SIT1 promoters was notably decreased in an stp1 stp2 deletion mutant, whereas the PHO84 and PHO89 gene reporters were unaffected. Our findings add a further layer of complexity to the regulation of ENA1 and suggest that the SPS pathway might participate in the regulation of a subset of alkali-inducible genes.


Assuntos
Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Aminoácidos/metabolismo , Regulação Fúngica da Expressão Gênica , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/metabolismo
4.
Int J Mol Sci ; 23(3)2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-35163251

RESUMO

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico/fisiologia , Fenótipo , Proteína Fosfatase 1/metabolismo
5.
Int J Mol Sci ; 21(20)2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33086699

RESUMO

The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.


Assuntos
Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/toxicidade , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/toxicidade , Saccharomyces cerevisiae/metabolismo , Temperatura Alta , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
7.
Antimicrob Agents Chemother ; 60(10): 6313-25, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27527084

RESUMO

Eradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms.


Assuntos
Antibacterianos/farmacologia , Proteína Catiônica de Eosinófilo/química , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Aglutinação/efeitos dos fármacos , Animais , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Plâncton/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologia
8.
FEBS Lett ; 596(12): 1556-1566, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35278214

RESUMO

Overexpression of Saccharomyces cerevisiae protein phosphatase Ppz1 strongly impairs cell growth. Ppz1 is negatively regulated by its subunit Hal3, and Hal3 overexpression fully counteracts the toxic effects derived from high levels of the phosphatase. We show that Ppz1 localizes at the plasma membrane, and that co-expression of Hal3 recruits Ppz1 to internal membranes (mostly vacuolar). This effect is not observed in a catalytically impaired mutant of Ppz1. Disruption of intracellular trafficking by deletion of the ESCRT-0 component VPS27 abolishes both Hal3-mediated relocalization of Ppz1 and normalization of cell growth, without affecting Ppz1 protein levels. We propose that Hal3 counteracts the toxic effects caused by excess of Ppz1 not only by inhibiting its enzymatic activity but also by recruiting the phosphatase to internal structures.


Assuntos
Proteínas de Ciclo Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Fosfoproteínas Fosfatases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
FEBS J ; 289(23): 7500-7518, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35811492

RESUMO

The function of the Saccharomyces cerevisiae Ppz1 phosphatase is controlled by its inhibitory subunit Hal3. Hal3 is a moonlighting protein, which associates with Cab3 to form a decarboxylase involved in the CoA biosynthetic pathway. Hal3 is composed by a conserved core PD region, required for both Ppz1 regulation and CoA biosynthesis, a long N-terminal extension, and an acidic C-terminal tail. Cab3 has a similar structure, but it is not a Ppz1 inhibitor. We show here that deletion or specific mutations in a short region of the N-terminal extension of Hal3 compromise its function as a Ppz1 inhibitor in vivo and in vitro without negatively affecting its ability to interact with the phosphatase. This study defines a R-K-X(3) -VTFS- sequence whose presence explains the unexpected ability of Cab3 (but not Hal3) to regulate Ppz1 function in Candida albicans. This sequence is conserved in a subset of fungi and it could serve to estimate the relevance of Hal3 or Cab3 proteins in regulating fungal Ppz enzymes. We also show that the removal of the motif moderately affects both Ppz1 intracellular relocalization and counteraction of toxicity in cells overexpressing the phosphatase. Thus, our work contributes to our understanding of the regulation of Ppz phosphatases, which are determinants for virulence in some pathogenic fungi.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Monoéster Fosfórico Hidrolases , Coenzima A , Fosfoproteínas Fosfatases/genética , Proteínas de Saccharomyces cerevisiae/genética
10.
J Fungi (Basel) ; 7(12)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34946993

RESUMO

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

11.
Sci Rep ; 10(1): 15613, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973189

RESUMO

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.


Assuntos
Regulação Fúngica da Expressão Gênica , Metaboloma , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Ciclo Celular , Dano ao DNA , Fosfoproteínas Fosfatases/genética , Fosforilação , Espécies Reativas de Oxigênio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
12.
Eur J Med Chem ; 152: 590-599, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29763807

RESUMO

Eradication of established biofilm communities of pathogenic bacteria is one of the pending challenges in the development of new antimicrobial agents. In particular, the dreaded nosocomial Pseudomonas aeruginosa forms microbial communities that offer an enhanced resistance to conventional antibiotics. Recently, we have described an engineered antimicrobial peptide derived from the human RNase3, also named the eosinophil cationic protein (ECP), RN3 (5-36), which combines bactericidal activity with high cell agglutination and lipopolysaccharide (LPS) affinity. Through a single replacement scan library using the SPOT methodology we have evaluated both the contribution of sequence positioning and amino acid singularity towards the peptide biological and physicochemical properties. Results indicate that the ECP N-terminus has already been extensively improved through evolution to provide high antimicrobial activity; hence most substitutions improving its antimicrobial performance are in detriment of safety towards host tissues. Only three positions were identified, occupied by polar residues on the first α-helix of the protein and replaceable by a hydrophobic residue, allowing an extended N-terminal patch that mediates bacterial agglutination. Among the best candidates, an Ile replacement proved best in improving the peptide therapeutic window. The novel engineered peptides encompass both the LPS-binding and aggregation-prone regions of parental ECP, providing the appropriate structural features for peptide attachment to the bacterial exopolysaccharide layer and bacterial cell membrane destabilization, thereby promoting biofilm removal at micro molar concentrations. We conclude that the novel engineered peptides are promising lead candidates against Gram-negative biofilms.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteína Catiônica de Eosinófilo/antagonistas & inibidores , Biblioteca de Peptídeos , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína Catiônica de Eosinófilo/metabolismo , Células Hep G2 , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Estrutura Molecular , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade
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