Isabel Coutinho

New insights into cancer‐related proteins provided by the yeast model

Cancer is a devastating disease with a profound impact on society. In recent years, yeast has provided a valuable contribution with respect to uncovering the molecular mechanisms underlying this disease, allowing the identification of new targets and novel therapeutic opportunities. Indeed, several attributes make yeast an ideal model system for the study of human diseases. It combines a high level of conservation between its cellular processes and those of mammalian cells, with advantages such as a short generation time, ease of genetic manipulation and a wealth of experimental tools for genome‐ and proteome‐wide analyses. Additionally, the heterologous expression of disease‐causing proteins in yeast has been successfully used to gain an understanding of the functions of these proteins and also to provide clues about the mechanisms of disease progression. Yeast research performed in recent years has demonstrated the tremendous potential of this model system, especially with the validation of findings obtained with yeast in more physiologically relevant models. The present review covers the major aspects of the most recent developments in the yeast research area with respect to cancer. It summarizes our current knowledge on yeast as a cellular model for investigating the molecular mechanisms of action of the major cancer‐related proteins that, even without yeast orthologues, still recapitulate in yeast some of the key aspects of this cellular pathology. Moreover, the most recent contributions of yeast genetics and high‐throughput screening technologies that aim to identify some of the potential causes underpinning this disorder, as well as discover new therapeutic agents, are discussed.

Aspartic vinyl sulfones: inhibitors of a caspase-3-dependent pathway

In this article we describe an expanded structure-activity relationship study for vinyl sulfones as caspase-3 inhibitors, a topic virtually unexplored in the existing literature. Most remarkably, and to our surprise, tripeptidyl vinyl sulfones were not active for caspase-3, opposite to other examples described in literature for peptidyl vinyl sulfones as potent cysteine protease inhibitors of clan CA. Moreover, the caspase-3 inhibitory activity of vinyl sulfones using an in vitro assay was then confirmed using a yeast cell-based assay. The results show that Fmoc-protected vinyl sulfones containing only the Asp moiety are inhibitors of a caspase-3-dependent pathway and the IC50 values obtained in the yeast assay are in the same order of magnitude of that obtained with the caspase-3 inhibitor tetrapeptidyl chloromethyl ketone, Ac-DEVD-CMK. This observation is consistent with appropriate cell permeability properties displayed by the vinyl sulfone inhibitors, as reflected by logP values ranging from 1.1 to 3.4. Overall, these results suggest that vinyl sulfones containing Asp at P1 should be considered for further optimization as caspase inhibitors and modulators of caspase-3-dependent pathways.

Distinct regulation of p53-mediated apoptosis by protein kinase Cα, δ, ε and ζ: Evidence in yeast for transcription-dependent and -independent p53 apoptotic mechanisms

The role of individual protein kinase C (PKC) isoforms in the regulation of p53-mediated apoptosis is still uncertain. Using yeast cells co-expressing the human wild-type p53 and a single mammalian PKCα, δ, ε or ζ, we showed a differential regulation of p53-mediated apoptosis by these PKC isoforms. Whereas PKCα and ζ had no effect on p53 activity, PKCδ and ε stimulated a p53-mediated mitochondria-dependent apoptosis. Moreover, using pifithrin-α and -μ, selective inhibitors of p53 transcriptional activity and mitochondrial p53 translocation, respectively, we showed the activation of a transcription-dependent and -independent p53-mediated apoptosis by PKCδ and ε. The activation of mitochondrial p53 translocation by PKCδ and ε was further confirmed by immunofluorescence and Western blot analysis. Together, this work reveals the conservation in yeast of functional transcription-dependent and -independent p53 apoptotic mechanisms. Furthermore, it gives mechanistic insights about the regulation of p53-mediated apoptosis by PKCδ and ε through modulation of p53 transcriptional activity and of its translocation to mitochondria. Finally, it underscores a major role of PKCδ and ε as positive regulators of p53-mediated apoptosis, and therefore as promising therapeutic targets in cancer.

Differential regulation of p53 function by protein kinase C isoforms revealed by a yeast cell system

The complexity of the mammalian p53 pathway and protein kinase C (PKC) family has hampered the discrimination of the effect of PKC isoforms on p53 activity. Using yeasts co‐expressing the human wild‐type p53 and a mammalian PKC‐α, ‐δ, ‐ε or ‐ζ, we showed a differential regulation of p53 activity and phosphorylation state by PKC isoforms. Whereas PKC‐α reduced the p53‐induced yeast growth inhibition and cell cycle arrest, PKC‐δ and ‐ε enhanced the p53 activity through p53 phosphorylation, and PKC‐ζ had no effect on p53. This work identified positive and negative p53 regulators which represent promising pharmacological targets in anti‐cancer therapy.

Selective activation of protein kinase C-δ and -ɛ by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U)

6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC-alpha and -betaI), novel (nPKC-delta and -epsilon) and atypical (aPKC-zeta), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC, coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs. The isoform-selectivity of coleon U for nPKC-delta and -epsilon was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase- and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC, recognised as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC-delta and -epsilon activation, it represents a promising compound for evaluation as an anti-cancer drug.

Rapid method for testing the susceptibility of Aspergillus fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole by assessment of oxygen consumption

OBJECTIVES Antifungal stress conditions affect fungal germination and growth. The assessment of oxygen consumption resulting from the challenge of Aspergillus fumigatus conidia with antifungal agents might be predictive of the susceptibility of this species to the agents evaluated. METHODS The antifungal susceptibilities of A. fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole were evaluated for 20 clinical strains by two methods: the rapid assessment of oxygen consumption and the CLSI M38-A2 microdilution method. For the determination of oxygen consumption, conidia were suspended in RPMI 1640 medium with two different concentrations of each antifungal drug (0.25 and 2 mg/L); the oxygen consumption was quantified in a biological oxygen monitor. RESULTS A. fumigatus strains showed a wide spectrum of amphotericin B, itraconazole and voriconazole MICs (0.06 to >16 mg/L), but posaconazole MICs ranged from 0.06 to 1 mg/L. Distinct respiratory kinetics, which corresponded to the MIC results, were found. Strains with the highest itraconazole and voriconazole MICs grew faster, undoubtedly consuming the oxygen available in the liquid medium. The reproducibility of this new method was adequate (87%), as well as the agreement with the CLSI method (85%). CONCLUSIONS Although the potential of this new and rapid method (4-8 versus 48 h CLSI method) for evaluating the susceptibility of A. fumigatus to the antifungal agents has been demonstrated by these preliminary results, further collaborative studies with more isolates should better assess the value of this methodology for testing isolates in the clinical laboratory.

Potential small-molecule activators of caspase-7 identified using yeast-based caspase-3 and -7 screening assays

Caspases-3 and -7 are at the core of the execution phase of apoptosis. The search for activators of these proteases has therefore deserved particular attention in the field of anticancer drug discovery. Here, a simplified yeast-based screening approach was developed and used to search for activators of caspases-3 and -7, followed by evaluation of the activity of the selected compounds in the human tumor cell lines HL-60 (acute promyelocytic leukemia) and MCF-7 (breast adenocarcinoma). By using the yeast approach, two potential activators of caspase-7, 5,6-dihydroxy-7-prenyloxyflavone (1a) and 3-hydroxy-7-geranyloxyflavone (2a), were identified. Unlike the known caspases-3 and -7 activator, the procaspase activating compound-1 (PAC-1), these flavonoids did not interfere with the caspase-3 activity in yeast. Moreover, flavonoids 1a and 2a processed procaspase-7 to the active caspase-7 both in yeast and in vitro processing assays, and inhibited the growth of HL-60 and MCF-7 human tumor cells with higher potencies than PAC-1, particularly in the absence of caspase-3 (MCF-7 cells). In MCF-7 cells, the flavonoids processed procaspase-7, increased its activity and sensitized these cells to the effects of the cytotoxic drug, etoposide. In conclusion, the developed yeast target-based screening assays led to the identification of potential caspase-7 activators. A proof of concept is therefore provided for the effectiveness of the yeast assays in the discovery of caspase activators. Additionally, the identified compounds may pave the way for a new class of caspase activators with improved anticancer properties.

Yeast as a Powerful Model System for the Study of Apoptosis Regulation by Protein Kinase C Isoforms

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of signals that control different cellular processes, such as cell death and proliferation. This family comprises at least 10 isoforms that regulate apoptosis in an isoformspecific manner. However, controversial data about the role of individual PKC isoforms in apoptosis regulation are frequently reported. The co-existence of several PKC isoforms in a same mammalian cell, the distinct expression profile of PKC isoforms in different cell types, and the different stimulus applied may explain such contradicting results. Therefore major advances in the understanding of the molecular mechanisms that regulate the function of PKC isoforms in apoptosis are still required. Yeast has proved to be a valuable research tool to investigate molecular aspects of apoptosis regulation. Additionally, the conservation in yeast of major functional and molecular properties of mammalian PKC isoforms favours the use of this simpler cell model to uncover relevant aspects of apoptosis regulation by this kinase family. In this review, we cover the current knowledge about the role of different PKC isoforms in apoptosis. Moreover, we discuss the contribution of yeast to unravel several controversial issues about apoptosis regulation by PKC isoforms. The exploitation of yeast cells expressing individual PKC isoforms towards the identification of isoform-specific PKC modulators is also discussed. The studies here summarised highlight that the yeast cell model system can provide valuable insights in the PKC research field.