In addition to apoptosis, metacapases function to regulate various other processes that promote and sustain life. For example, the Saccharomyces cerevisiae metacaspase Yca1 promotes cellular fitness by regulating insoluble protein levels. However, the mechanism(s) that regulate this proteostasis function for Yca1 have remained elusive. Here, using proteomics coupled to protein interaction studies, we describe a role for Yca1 in restraining deposition to the insoluble proteome and further identify a post-translational regulatory mechanism for modulating Yca1 function. Our initial analyses uncovered a role for Yca1 in aggregate assembly where Yca1, in coordination with the Cdc48 chaperone, regulates the composition of the insoluble proteome. Interestingly, loss of Yca1 was correlated with reduced sequestration of proteins related to ribosomal and translational processes in the insoluble protein fraction during heat stress. Subsequent proteomic analyses identified a regulatory mechanism for Yca1 mediated by the ubiquitin system, a feature that was instrumental for limiting insoluble protein content. Specifically, we noted K355 ubiquitination and S346 phosphorylation as key modifications that directed Yca1 function to maintain proteostasis. Loss of function mutations at these sites led to increased retention of insoluble protein and increased vacuolar structures. Surprisingly, loss of Yca1 also affected ubiquitin homeostasis in vivo as observed by reduced levels of low molecular weight free ubiquitin. Upon further analysis, we observed that the ubiquitin precursor protein Rsp31 was cleaved by Yca1 suggesting a possible role for Yca1 in de novo ubiquitin synthesis. Together, these analyses suggest that post-translational modifications of Yca1 are critical regulatory features for this protease, and that this enzyme regulates cell proteostasis in combination with other chaperone and protein degradation machinery.

Plasmodium falciparum est un protozoaire parasite responsable du paludisme causant la mort d’environ un million de personnes par an. La résistance médicamenteuse du parasite augmente la pathogénicité de cette maladie. Il est question ici d’explorer les mécanismes moléculaires impliqués dans la mort cellulaire programmée (apoptose) du parasite en présence de chloroquine (CQ) et de tester l’hypothèse qu’une résistance à la CQ peut s’expliquer en partie par une défaillance de ce mécanisme de mort. Dans un premier temps l’étude des marqueurs de l’apoptose (TUNEL, JC1, formes pyknotiques) montre qu’une souche sensible de parasite (3D7) à la CQ peut subir une apoptose en présence de CQ alors qu’une souche résistante (7G8) présente un défaut d’apoptose. Dans un deuxième temps nous montrons que la protéine PfMCA1 (P. falciparum métacaspase 1) présente une structure et une maturation protéolytique proche de celui des caspases faisant de cette protéine un candidat potentiellement impliqué dans l’apoptose du parasite. Dans un troisième temps nous montrons que l’expression du domaine catalytique de PfMCA1 dans la levure induit une mort cellulaire et un retard de croissance de la levure. Nous montrons également que PfMCA1 présente une activité enzymatique de type arginase alors que les effets induit par sa surexpression peuvent être inhibés par l’ajout d’un inhibiteur de protéases spécifiques des aspartates. Ces résultats suggèrent que PfMCA1 pourrait agir comme une protéine initiatrice induisant l’action d’une protéase effectrice spécifique des aspartates conduisant à la mort cellulaire. Cette hypothèse testée chez la levure reste à confirmée chez P. falciparum / Plasmodium falciparum is a protozoan parasite responsible for malaria causing one million deaths per year. Drug resistance of the parasite increases the pathogenicity of this disease. In this thesis, it is question to explore the molecular pathway involved in programmed cell death (apoptosis) of the parasite in the presence of chloroquine (CQ) and to test the hypothesis that CQ resistance could be partly explained by a failure of such a mechanism. In a first step, we showed that a sensitive clone (3D7) exhibited the classical hallmarks of apoptosis (DNA fragmentation, mitochondrial depolarization) under a CQ pressure while a resistance clone failed to undergo apoptosis. In a second step we show that the protein PfMCA1 (P. falciparum metacaspase 1) has a structure and a processing similar to the well known caspases which are the key effectors of apoptosis for higher eukaryotic cells. In a third step we show that expression of the catalytic domain of PfMCA1 in yeast induces cell death and growth retardation of yeast. We show that PfMCA1 presented an arginine-specific protease activity while the effects induced by its overexpression were inhibited by an aspartate-specific protease inhibitor (z-VAD-fmk). These results suggest that PfMCA1 might act as an initiator protein inducing an aspartate-specific protease effector leading to cell death. This hypothesis tested in yeast remains to be confirmed in P. falciparum

Programmed cell death (PCD) refers to a genetically controlled process causing the death of certain cells or tissues. In plants PCD is critical in normal development of for instance xylem vessels. A group of proteins called metacaspases are believed to play a pivotal role in PCD in plants. As Metacaspase 9 have been shown to be upregulated in Populus during xylem maturation this study attempted to identify genes affecting its expression in Arabidopsis thaliana by forward genetics using a reporter line with GFP fused to the promoter of Metacaspase 9 (AtMC9). Ethyl methanesulfonate seed mutagenesis was used to generate mutants resulting in eleven mutant lineages with a GFP expression pattern deviating from that of the reporter line. These mutants fell into two categories; low/no-signal mutants and ectopic expressors. Several of the low/no-signal mutants had longer roots at five to eight days after germination, a time point shown to be critical for metaxylem differentiation. Further studies of their roots would reveal whether the developing xylem is abnormal or not. Deep sequencing provided evidence for involvement of abscisic acid and polyamines in regulation of AtMC9 expression. Sequencing from a low/no-signal mutant suggests that AtMC9 expression might be affected also by disturbed lignin biosynthesis. Rescuing mutant lineages through transformations with fully functional forms of the candidate genes is the next step to experimentally validate that the candidate genes are involved in the observed changes in AtMC9 expression in each of the isolated mutants.

Programmed cell death (PCD) is an indispensible process in plant and animal systems that serves to eliminate cells and/or tissues and recycle nutrients from these tissues to the rest of the organism. In animals, PCD is referred to as apoptosis and is performed by caspases, a family of aspartate-specific cysteine proteinases that serve to perceive the cell death signal and execute the cell death phenotype. In 2000, Uren et al. discovered a new family of cysteine proteinases in plants called metacaspases – distant arginine/lysine-specific relatives of animal caspases – thought to be involved in plant PCD. The goal of this study was to correlate SlMCA2 expression with PCD in tomato. Polyclonal antibodies were obtained against the Type I metacaspase SlMCA2 in Solanum lycopersicum and used for Western blot analyses. BY-2 cell biolistics and in-situ hybridization were used to investigate where SlMCA2 protein and mRNA accumulate in various tissues. Results produced were replicated a minimum of three times and correlate SlMCA2 to PCD, but not initiation of PCD.

Xylem is an inherent feature of all vascular plants and functions in water transport and mechanical support. In order to efficiently transport water, xylem cells are reinforced by secondary walls before they undergo programmed cell death and their cell contents are removed by autolysis to create a hollow tube. During their differentiation, xylem cells express various hydrolytic enzymes, such as proteases, nucleases and lipases, but only in a few examples has their role in xylem cell death been characterized. This thesis focuses on the regulatory aspects of xylem cell death and the autolytic cell clearance in vessel elements and fibers of hybrid aspen (Populus tremula L. x tremuloides Michx.) and in vessel elements of Arabidopsis thaliana. Using comparative transcriptomic analysis, candidate genes for fiber-specific cell death processes were identified. Further, a hypothesis is presented on the regulation of thermospermine levels in the vasculature by a negative feedback-loop involving auxin and the class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factor HOMEOBOX8 (PtHB8). The role of the Arabidopsis METACASPASE9 (AtMC9) in xylem cell death was characterized using molecular tools, such as reporter lines and fluorescent fusion proteins, and electron microscopy (TEM). This showed that cell death initiation is not controlled by AtMC9. Instead, evidence is presented for the involvement of AtMC9 in the post mortem autolysis of vessel elements that follows tonoplast rupture and leads to the formation of the hollow conduit. Cell death-associated genes were further observed to be expressed during the emergence of lateral roots in Arabidopsis thaliana. This led to the discovery that cells overlying a lateral root primordium undergo cell death, which was demonstrated by detection of DNA degradation and TEM analysis. It is concluded that cell death facilitates emergence of lateral roots through the overlying tissues in a concerted manner with cell wall remodelling. Together, these findings show that although individual hydrolytic enzymes may be dispensable for plant growth and development, their common regulators are the tool for understanding their function and importance. / Xylem är en karakteristisk vävnad i alla kärlväxter som leder vatten och mineraler samt har mekanisk stödfunktion. För att effektivt kunna transportera vatten förstärks xylemceller med sekundära cellväggar innan de dör genom programmerad celldöd. Deras cellinnehåll bryts ner genom autolys för att skapa ett ihåligt rör. Xylemceller uttrycker under sin differentiering olika hydrolytiska enzymer, såsom proteaser, lipaser och nukleaser, men bara för ett fåtal av dessa har funktionen under xylemcelldöd kartlagts. Denna avhandling fokuserar på reglering av xylemcelldöden och den autolytiska nedbrytningen av cellen, i såväl kärlelement och fibrer av hybridasp (Populus tremula L. x tremuloides Michx.) som i kärlelement av backtrav (Arabidopsis thaliana). Med hjälp av jämförande transkriptomanalys identifierades kandidatgener för fiber-specifika celldödsprocesser i hybridasp. Vidare utvecklades en hypotes om reglering av termosperminnivåer i vaskulaturen genom en negativ feedback-loop, som omfattar auxin reglering och klass III homeodomän-leucinzipper (HD-ZIP III) transkriptionsfaktorn HOMEOBOX8 (PtHB8). Funktionen av Arabidopsis METACASPASE9 (AtMC9) under xylemcelldöd karakteriserades med molekylära verktyg, såsom reporterlinjer och fluorescerande fusionsproteiner och elektronmikroskopi (TEM). Dessa analyser visade att celldödens initiering inte styrs av AtMC9. Istället presenteras bevis för en roll av AtMC9 i autolysen av kärlelement som sker post mortem efter att vakuolen har gått sönder och som slutför bildandet av det tomma kärlet. Genuttryck som associeras med celldöd observerades också under utvecklingen av laterala rötter i Arabidopsis thaliana. Detta ledde till upptäckten att celler som ligger ovanför ett lateralrotprimordium dör en programmerad celldöd och visar tecken på DNA-nedbrytning och autolys i TEM-analyser. Slutsatsen av denna studie är att celldöd i samspel med cellväggsmodifiering underlättar utväxten av laterala rötter genom de överliggande cellagren. Sammantaget tyder dessa upptäckter på att även om enstaka hydrolyserande enzymer inte är nödvändiga för växternas tillväxt och utveckling, så kan deras gemensamma reglering nyttjas för att förstå deras funktion och betydelse.

The evolutionary success of land plants was fostered by the acquisition of the xylem vascular tissue which conducts water and minerals upwards from the roots. The xylem tissue of flowering plants is composed of three main types of cells: the sap-conducting tracheary elements (TE), the fibres which provide mechanical support and the parenchyma cells which provide metabolic support to the tissue. Both the TEs and the fibres deposit thick polysaccharidic secondary cell walls (SCWs), reinforced by a rigid phenolic polymer called lignin. The cell walls of TEs form efficient water conducting hollow tubes after the TEs have undergone programmed cell death (PCD) and complete protoplast degradation as a part of their differentiation. The work presented in this thesis studied the regulation of TE PCD by characterizing the function of the candidate PCD regulator METACASPASE 9 (MC9) in Arabidopsis thaliana xylogenic cell suspensions. These cell suspensions can be externally induced to differentiate into a mix of TEs and parenchymatic non-TE cells, thus representing an ideal system to study the cellular processes of TE PCD. In this system, TEs with reduced expression of MC9 were shown to have increased levels of autophagy and to trigger the ectopic death of the non-TE cells. The viability of the non-TE cells could be restored by down-regulating autophagy specifically in the TEs with reduced MC9 expression. Therefore, this work showed that MC9 must tightly regulate the level of autophagy during TE PCD in order to prevent the TEs from becoming harmful to the non-TEs. Hence, this work demonstrated the existence of a cellular cooperation between the TEs and the surrounding parenchymatic cells during TE PCD. The potential cooperation between the TEs and the neighbouring parenchyma during the biosynthesis of lignin was also investigated. The cupin domain containing protein PIRIN2 was found to regulate TE lignification in a non-cell autonomous manner in Arabidopsis thaliana. More precisely, PIRIN2 was shown to function as an antagonist of positive transcriptional regulators of lignin biosynthetic genes in xylem parenchyma cells. Part of the transcriptional regulation by PIRIN2 involves chromatin modifications, which represent a new type of regulation of lignin biosynthesis. Because xylem constitutes the wood in tree species, this newly discovered regulation of non-cell autonomous lignification represents a potential target to modify lignin biosynthesis in order to overcome the recalcitrance of the woody biomass for the production of biofuels.

Programmed cell death in animals, plants and protists is in part regulated by a variety of proteases, including cysteine aspartyl proteases, (caspases, paracaspases and metacaspases), cathepsins, subtilisin-like serine proteases, vacuolar processing enzymes and the proteasome. The role of different proteases in the cell death responses of the fungi is however largely unknown. A greater understanding of the fungal cell death machinery may provide new insights into the mechanisms and evolution of PCD and potentially reveal novel targets for a new generation of antifungal drugs. The role of a metacaspase encoding gene, MCA1, in the cell death response of the human pathogen Candida albicans pathogen has been investigated by functional analysis. MCA1 deletion not only alters the sensitivity of cells to a number of cell death stimuli, it also enhances virulence in an insect model. C. albicans shows altered cell and colony morphology on Lee’s medium. Evidence is presented to suggest that these functions appear to be dependent upon active mitochondria. In this study it has also been shown that key caspase substrates may be conserved between humans and the yeasts Saccharomyces cerevisiae and Candida albicans. Many substrates, particularly those which are essential, have retained their caspase cleavage motifs. 14 protease mutants displayed altered activity against caspase 1, 3, 6 or 8 substrates during acetic acid-induced PCD and caspase 1-like activity appeared to be particularly associated with PCD. Using a novel bioinformatic analysis of experimental LC-MS/MS data, changes in the degradation patterns of the proteome (destructome) following acetic acid-induced cell death have been investigated in wild-type yeast. In addition, potential native substrates of the yeast Mca1 have also been identified. The future challenge is to characterise the destructome of different proteases under a range of cell death conditions. In this way it may be possible to identify key components of the cell death machinery and their substrates and so reveal the most promising targets for future therapeutics.