Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules.

BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

Association of increased basic salivary proline-rich protein 1 levels in induced sputum with type 2-high asthma.

BACKGROUND: The aim of this study is to reveal whether basic salivary proline-rich protein BstNI subfamily 1 (PRB1) may be used as a diagnostic biomarker for type 2-high asthma. METHODS: PRB1 protein levels in the induced sputum of 67 subjects with asthma and 27 controls were determined by an enzyme-linked immunosorbent assay. Correlation analyses between PRB1 in the induced sputum and airway inflammatory indicators were also performed. RESULTS: PRB1 protein levels were significantly upregulated in the induced sputum of asthmatic patients (p =0.0098) and correlated with clinical eosinophil-related indicators and type 2 airway inflammation. These results indicate that PRB1 is a promising biomarker for type 2-high asthma. CONCLUSIONS: The expression of PRB1 in induced sputum is a potential biomarker for type 2-high asthma. The results of this study present new insights into the diagnosis and treatment of asthma.

Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast.

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a nonenzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize large quantities of polyP. In this study, we evaluated the polyphosphorylation status of over 200 candidate targets in Saccharomyces cerevisiae. We report eight new polyphosphorylated proteins that interact genetically and physically with previous targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising questions regarding the location of polyphosphorylation in vivo.

A Cinderella story: how the vacuolar proteases Pep4 and Prb1 do more than cleaning up the cell's mass degradation processes.

Recently, several research groups have assigned non-vacuolar functions to the well-known Saccharomyces cerevisiae vacuolar proteases Pep4 and Prb1, which are also known as proteinases A and B. These non-vacuolar activities seem to be autophagy-independent and stress-induced and suggest an unexplored but possibly prominent role for the proteases outside the vacuole. The functions range from the involvement in programmed cell death, to protection from hazardous protein forms and regulation of gene expression. We propose that a deeper understanding of these molecular processes will provide new insights that will be important for both fungal biology as well as studies in mammalian cells, as they might open up perspectives in the search for novel drug targets. To illustrate this, we summarize the recent literature on non-vacuolar Pep4 and Prb1 functions in S. cerevisiae and review the current data on the protein homologs in pathogenic fungi.

Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells.

In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm(5) ) and 5-methylcarboxymethyl (mcm(5) ) side chains on uridines present at the wobble position (U34 ) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5' AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.

Prb1, a subtilisin-like protease, is required for virulence and phenotypical traits in the chestnut blight fungus.

Subtilisin-like proteases are widely distributed and reported to be required for virulence in pathogenic fungi. In chestnut blight fungus Cryphonectria parasitica, prb1, encoding a putative subtilisin-like protease, was expressed and recombinant Prb1 protein was shown to have a protease activity in vitro. prb1-deleted mutants exhibited reduced total protease activity by 60%. The Δprb1 mutants showed a phenotype of reduced aerial hyphae, lower level of sporulation, and a significant reduction in virulence. Additionally, site-directed mutagenesis of Prb1 protein revealed that D195, H227, and S393 are critical for C. parasitica Prb1 function in vivo. Transcriptional analysis showed that deletion of prb1 also reduced the transcript accumulation levels for genes encoding key components of the heterotrimeric G-protein signaling pathway, including cpga1, cpgb1, cpgc1, and ste12. Furthermore, deletion of prb1 results in the accumulation of autophagic bodies in the fungus. Taken together, our results showed that prb1-encoded protease functions in the regulation of virulence, phenotypical traits, and autophagy in C. parasitica.

Perspectivas en la genómica del retinoblastoma: Implicaciones del gen supresor de tumor RB1 / Genomic retinoblastoma perspectives: Implications of supressor gene of tumor RB1

In order to define the molecular and cellular bases of the development of retinoblastomas it is necessary to know its etiology, and to apply the advances in genome technology to this kind of neoplasia. Retinoblastomas are childhood tumors of the eye with an average incidence of one case in every 15,000-20,000 live births, which occur in sporadic and hereditary forms. The sporadic form appears regularly as a unilateral tumor, while in the familial form of the disease, tumors may be unilateral and bilateral. This neoplasia is characterized by leukocoria, strabism, and heterochromia. The retinoblastoma gene (RBl) is a molecular marker of retinoblastoma tumors. This gene is located in chromosome 13q14.2 and encodes a nuclear phosphoprotein (pRB) of 110 KDa, which plays a major role in cell proliferation control through cell cycle-regulated phosphorylation/dephosphorylation cycles of this protein. The RBl gene is mainly affected by point mutations, which occur most frequently in exons 3, 8, 18 and 20. At the end of the last century, DNA technology has improved notably, allowing for its application to the study of a vast array of diseases. The aim of this work is to show the molecular aspects involved in retinoblastoma which are currently deciphering; this is possible thanks to new technology platforms that have been developed. This will allow us in a near future, to offer tests for the early diagnoses, prognoses, and the determination of individual predisposition towards this neoplasia.

El retinoblastoma es una neoplasia embrionaria que se manifiesta en dos formas: esporádica (no heredada) o familiar (heredada). En los casos esporádicos el tumor es unilateral y en la forma familiar puede presentarse de manera unilateral o bilateral. Esta neoplasia tiene una incidencia promedio de 1/15,000 nacidos vivos, presentando signos y síntomas que incluyen leucocoria, estrabismo, midriasis unilateral y heterocromía. El gen que predispone al desarrollo de retinoblastoma es RBl y se localiza en el cromosoma 13 en la región ql4.2. El gen RBl codifica para una fosfoproteína nuclear que participa de manera importante en la regulación del ciclo celular. De acuerdo con la hipótesis de Knudson, para que se desarrolle la neoplasia se deben presentar dos mutaciones en el gen RBl. Las mutaciones puntuales son las que más frecuentemente se presentan en el gen RBl; la mayoría de los estudios indican que los exones 3, 8, 18, 19 y 20 son las regiones de mutación preferencial. En la áltima década ha habido un gran avance en la tecnología del DNA, lo cual hace posible su aplicación en diferentes enfermedades. Estas herramientas moleculares podrían ser de gran utilidad en el diagnóstico o conocimiento de la predisposición a desarrollar un retinoblastoma. Entre estas valiosas herramientas se cuenta con la hibridación fluorescente realizada in situ, hibridación genómica comparativa, las microhileras y por áltimo la identificación de polimorfismos de un sólo nucleótido. En conclusión, actualmente se están descifrando los aspectos moleculares que están relacionados con el retinoblastoma, gracias a la aplicación de nuevas plataformas tecnológicas. Esto permitirá en un futuro próximo ofrecer pruebas para un diagnóstico temprano o para conocer el pronóstico y la predisposición de individuos a desarrollar esta patología. Con el fin de entender las bases celulares y moleculares del desarrollo del retinoblastoma, el objetivo del presente trabajo es mostrar el estado del arte del conocimiento de esta neoplasia, así como su origen y los avances en la genómica aplicada al retinoblastoma.