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Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion (~75%) or mutation (~10%) of the ubiquitin E3 ligase A (UBE3A) gene, which encodes a HECT type E3 ubiquitin protein ligase. Although the critical substrates of UBE3A are unknown, previous studies have suggested a critical role of nuclear UBE3A in AS pathophysiology. Here, we investigated to what extent UBE3A missense mutations disrupt UBE3A subcellular localization as well as catalytic activity, stability and protein folding. Our functional screen of 31 UBE3A missense mutants revealed that UBE3A mislocalization is the predominant cause of UBE3A dysfunction, accounting for 55% of the UBE3A mutations tested. The second major cause (29%) is a loss of E3-ubiquitin ligase activity, as assessed in an Escherichia coli in vivo ubiquitination assay. Mutations affecting catalytic activity are found not only in the catalytic HECT domain, but also in the N-terminal half of UBE3A, suggesting an important contribution of this N-terminal region to its catalytic potential. Together, our results show that loss of nuclear UBE3A E3 ligase activity is the predominant cause of UBE3A-linked AS. Moreover, our functional analysis screen allows rapid assessment of the pathogenicity of novel UBE3A missense variants which will be of particular importance when treatments for AS become available.

The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells towards osteoblast and myocyte phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation. For these studies pre-adipocytes isolated from mouse adipose tissue and the 3 T3-L1 (pre)adipocyte cell line were applied. We performed FHL2 gain of function and knockdown experiments followed by extensive RNAseq analyses and phenotypic characterization of the cells by oil-red O (ORO) lipid staining. Through affinity-purification mass spectrometry (AP-MS) novel FHL2 interacting proteins were identified. Here we report that FHL2 is expressed in pre-adipocytes and for accurate adipocyte differentiation, this protein needs to be downregulated during the early stages of adipogenesis. More specifically, constitutive overexpression of FHL2 drastically inhibits adipocyte differentiation in 3 T3-L1 cells, which was demonstrated by suppressed activation of the adipogenic gene expression program as shown by RNAseq analyses, and diminished lipid accumulation. Analysis of the protein-protein interactions mediating this repressive activity of FHL2 on adipogenesis revealed the interaction of FHL2 with the Nuclear factor of activated T-cells 5 (NFAT5). NFAT5 is an established inhibitor of adipocyte differentiation and its knockdown rescued the inhibitory effect of FHL2 overexpression on 3 T3-L1 differentiation, indicating that these proteins act cooperatively. We present a new regulatory function of FHL2 in early adipocyte differentiation and revealed that FHL2-mediated inhibition of pre-adipocyte differentiation is dependent on its interaction with NFAT5. FHL2 expression increases with aging, which may affect mesenchymal stem cell differentiation, more specifically inhibit adipocyte differentiation.

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex post-translational modifications in a cellular setting.

Src-homology 3 (SH3) domains are small protein-protein interaction modules. While most SH3 domains bind to proline-x-x-proline (PxxP) containing motifs in their binding partners, some SH3 domains recognize motifs other than proline-based sequences. Recently, we showed that the SH3 domain of Candida albicans Rvs167-3 binds peptides enriched in hydrophobic residues and containing a single proline residue (RΦxΦxΦP, where x is any amino acid and Φ is a hydrophobic residue). Here, we demonstrate that the proline in this motif is not required for Rvs167-3 SH3 recognition. Through mutagenesis studies we show that binding of the peptide ligand involves the conserved tryptophan in the canonical PxxP binding pocket as well as residues in the extended n-Src loop of Rvs167-3 SH3. Our studies establish a novel, proline-independent, binding sequence for Rvs167-3 SH3 (RΦxΦxΦ) that is comprised of a positively charged residue (arginine) and three hydrophobic residues.

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid β-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae ; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C 2 carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid β-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid β-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans . Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid β-oxidation pathway. Here, we asked whether fatty acid β-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Δ/pex5Δ mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Δ/fox2Δ mutant, which lacks the second enzyme of the β-oxidation pathway. Both mutant strains had strongly reduced β-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Δ/fox2Δ mutant, and not the pex5Δ/pex5Δ mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Δ/fox2Δ mutant was comparable to that of the icl1Δ/icl1Δ mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid β-oxidation is not essential for virulence of C. albicans , implying that the attenuated virulence of the fox2Δ/fox2Δ mutant is largely due to a dysfunctional glyoxylate cycle.

In trypanosomes, MRP1 and MRP2 (previously called gBPx and gBPy, in which x and y indicate the MW of these proteins in a particular species) are guide (g)RNA binding proteins that are part of a large heteromeric complex that may play a role in U-insertion/deletion editing of mitochondrial mRNAs. In order to shed more light on the function of these proteins, we generated procyclic Trypanosoma brucei cell lines in which the levels of MRP 1 and/or MRP2 mRNA were downregulated by RNA interference (RNAi). Here we report that the RNAi-mediated knockdown of MRP1 and/or MRP2 resulted in severe growth inhibition and loss of both proteins. This loss occurred even in cells in which only one of the MRPs was targeted by RNAi, indicating a mutual dependence for stability of these proteins. The elimination of the MRPs substantially reduced the levels of edited cytB and RPS12 mRNAs, but resulted in little or no reduction of edited cox2, cox3 and A6 mRNAs, as measured in poisoned primer extension analyses. Surprisingly, we found a five-fold increase in ND7 mRNA editing in MRP1+2 double knockdown cells. In addition, the knockdowns also resulted in reduction in the amounts of mRNAs that do not undergo RNA editing (cox1, ND4 and ND5 mRNAs), but little change was observed for mitoribosomal 12S rRNA. Together, the results indicate that in procyclic T. brucei, MRP1 and MRP2 play a role in transcript-specific editing and other RNA processing activities.

RNA editing in mitochondria of kinetoplastid protozoa involves the posttranscriptional insertion and deletion of uridylate residues in protein encoding regions of pre-mRNAs. Editing is required to remove gene-encoded translational defects or to convert a nonsense sequence into a sense message. In cultured trypanosomatids, however, translationally defective pre-mRNAs for a number of NADH-dehydrogenase subunits are not converted into functional mRNAs by editing. In this report, the available data are discussed in the context of current models for RNA editing.

Transport of acetyl-CoA between intracellular compartments is mediated by carnitine acetyltransferases (Cats) that reversibly link acetyl units to the carrier molecule carnitine. The genome of the opportunistic pathogenic yeast Candida albicans encodes several (putative) Cats: the peroxisomal and mitochondrial Cat2 isoenzymes encoded by a single gene and the carnitine acetyltransferase homologs Yat1 and Yat2. To determine the contributions of the individual Cats, various carnitine acetyltransferase mutant strains were constructed and subjected to phenotypic and biochemical analyses on different carbon sources. We show that mitochondrial Cat2 is required for the intramitochondrial conversion of acetylcarnitine to acetyl-CoA, which is essential for a functional tricarboxylic acid cycle during growth on oleate, acetate, ethanol, and citrate. Yat1 is cytosolic and contributes to acetyl-CoA transport from the cytosol during growth on ethanol or acetate, but its activity is not required for growth on oleate. Yat2 is also cytosolic, but we were unable to attribute any function to this enzyme. Surprisingly, peroxisomal Cat2 is essential neither for export of acetyl units during growth on oleate nor for the import of acetyl units during growth on acetate or ethanol. Oxidation of fatty acids still takes place in the absence of peroxisomal Cat2, but biomass formation is absent, and the strain displays a growth delay on acetate and ethanol that can be partially rescued by the addition of carnitine. Based on our results, we present a model for the intracellular flow of acetyl units under various growth conditions and the roles of each of the Cats in this process.

The glyoxylate cycle, a metabolic pathway required for generating C-4 units from C-2 compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5 Delta/Delta strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2 Delta/Delta strain that is completely deficient in fatty acid beta-oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2 Delta/Delta strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2 Delta/Delta strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2 Delta/Delta mutant

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth inhibition. It also resulted in the loss of both proteins, even when only one of the MRP mRNAs was reduced, indicating a mutual dependence for stability. Elimination of the MRPs gave rise to substantially reduced levels of edited CyB and RPS12 mRNAs but little or no reduction of the level of edited Cox2, Cox3, and A6 mRNAs as measured by poisoned primer extension analyses. In contrast, edited NADH-dehydrogenase (ND) subunit 7 mRNA was increased 5-fold in MRP1+2 double knock-down cells. Furthermore, MRP elimination resulted in reduced levels of Cox1, ND4, and ND5 mRNAs, which are never edited, whereas mitoribosomal 12 S rRNA levels were not affected. These data indicate that MRP1 and MRP2 are not essential for RNA editing per se but, rather, play a regulatory role in the editing of specific transcripts and other RNA processing activities.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive ‘cut-and-paste’ reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of ∼10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3′-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

The nucleotide sequence of a 2.5-kb segment of the maxi-circle of Trypanosoma brucei mtDNA has been determined. The segment contains the gene for apocytochrome b, which displays about 25% homology at the amino acid level to the apocytochrome b gene from fungal and mammalian mtDNAs. Northern blot and S1 nuclease analyses have yielded accurate map positions of an RNA species in an area that coincides with the reading frame. The segment also contains two pairs of overlapping unassigned reading frames, which lack homology with any known mitochondrial gene or URF. The DNA sequence in these areas is AG-rich (70%), resulting in URFs with an unusually high level of glycine and charged amino acids (60%). They may not encode proteins, in spite of their size and the fact that abundant transcripts are mapped in these areas.