Deciphering the Kynurenine-3-Monooxygenase Interactome

Kynurenine-3-monooxygenase (KMO) is a mitochondrial enzyme in the kynurenine pathway (KP) through which tryptophan is degraded to NAD+. The central KP is altered in neurodegenerative diseases and other CNS disorders. The causative role of KP metabolites has been particularly well studied in the neurodegenerative disorder Huntington’s disease (HD), a fatal adult onset condition inherited in an autosomal dominant manner. In HD, flux in the KP is perturbed such that neurotoxic metabolites (3-hydroxykynurenine and quinolinic acid) of the pathway are increased relative to a neuroprotective metabolite (kynurenic acid). KMO lies at a critical branching point in the KP such that inhibition of KMO activity ameliorates this metabolic perturbation. Consequently, several recent studies have found that KMO inhibition is protective in models of HD. These findings have widespread implications in treating several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease where the KP is implicated in pathogenesis. The focus of this project was to better understand the cellular role(s) and interactions of KMO. To this end, a novel membrane yeast two hybrid approach was established and optimised to identify protein interaction partners for outer mitochondrial membrane proteins. This approach was implemented to identify protein interaction partners of human KMO and its yeast orthologue Bna4, which were confirmed by biochemical approaches. Additionally, genetic interaction partners of BNA4 identified by systematic genetic screens were individually validated by classic genetic manipulations. Bioinformatic tools were then used to identify enriched interaction networks for KMO using this novel interaction data. These analyses suggested possible roles for KMO in many processes, including energy metabolism, cytoskeleton organisation and response to infection and inflammation, providing evidence that KMO plays roles in diverse cellular pathways in addition to the KP

Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in Candida albicans

Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker’s yeast.The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively). Candida albicansURA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner

Origin replication complex binding, nucleosome depletion patterns, and a primary sequence motif can predict origins of replication in a genome with epigenetic centromeres

Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A “mini-ARS screen” identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion

Neocentromeres Provide Chromosome Segregation Accuracy and Centromere Clustering to Multiple Loci along a <i>Candida albicans</i> Chromosome

<div><p>Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble <i>de novo</i>, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used <i>Candida albicans</i>, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.</p></div

Neocentromere formation results in epigenetic activation of centromere clustering.

<p>Red lines mark centromeres. Green lines indicate neocentromere positions. Black diamond indicates the viewpoint for the plotted interaction profiles. A. Virtual 4C plots from the 10kb sequence surrounding the 4.5kb neocentromere region showing log-scaled Hi-C contact counts for all <i>C</i>. <i>albicans</i> chromosomes in the wild type (non-neocentromere) strain. B. Virtual 4C plots from the 10kb sequence surrounding the 166kb neocentromere region showing log-scaled Hi-C contact counts for all <i>C</i>. <i>albicans</i> chromosomes in the wild type (non-neocentromere) strain. C. Virtual 4C plots from the 10kb sequence surrounding the 4.5kb neocentromere region showing log-scaled Hi-C contact counts for all <i>C</i>. <i>albicans</i> chromosomes in YJB10777 (4.5kb neocentromere) strain. D. Virtual 4C plots from the 10kb sequence surrounding the 166kb neocentromere region showing log-scaled Hi-C contact counts for all <i>C</i>. <i>albicans</i> chromosomes in YJB10780 (166kb neocentromere) strain.</p

Transcriptional activity is repressed following neocentromere formation.

<p>Homozygous neocentromere strains YJB12027 (800kb center), YJB12028 (72.5kb center), and JYB12330 (826.5kb center) were grown in YPAD for 4 h. mRNA levels for <i>ORF19</i>.<i>951</i> (A), <i>ORF19</i>.<i>6668</i> (B), <i>ORF19</i>.<i>1121</i> (C) and <i>ORF19</i>.<i>949</i> (D) relative to the reference gene <i>TEF1</i> were measured by qRT-PCR. Data shown are mean ± SEM of 3 biological replicates. * p<0.01 by ANOVA and Tukey post-tests.</p

Neocentromere chromosome loss rate correlates with transcriptional activity, but not chromosomal position.

<p>A. The fold-difference in <i>URA3</i> loss rate between the mean rate for the native centromere strain and the mean rate of each neocentromere strain was plotted as a function of the fraction of the neocentromere CENP-A bound region that includes ORFs multiplied by the RNA-seq transcriptional measurement on a log2 scale of RPKM. Correlation between these two variables was high (r² = 0.71). B. The fold-difference in <i>URA3</i> loss rate between the mean rate for the native centromere strain and the mean rate of each neocentromere strain was plotted as a function of the distance between the neocentromere position and the native centromere. Correlation between these two variables was very low (r² = 0.06).</p

Neocentromere strains have different <i>URA3</i> loss rates.

<p>A. Fluctuation analysis of loss of <i>URA3</i> in control (<i>INT1/int1Δ</i>::<i>ura3</i>) (dark purple) and neocentromere <i>(CEN5/cen5Δ</i>::<i>ura3</i>) (magenta) strains. Cultures of each strain were grown in YPAD for 24 h at 30°C. Loss of <i>URA3</i> was quantified by plating cells on non-selective media (YPAD) and on media containing 5-FOA to select for loss of <i>URA3</i>. Colony counts were used to calculate the rate of loss per cell division. Results are the mean ± SEM of the rates calculated from at least 3 experiments, each with 8 cultures per condition. p<0.01 for strain differences by ANOVA. B. Cultures of each strain were grown in YPAD for 24 h at 30°C (magenta) or 39°C (purple). Loss of <i>URA3</i> was quantified by plating cells on non-selective media and on media containing 5-FOA to select for loss of <i>URA3</i>. Colony counts were used to calculate the rate of loss per cell division. Results are the mean ± SEM of the rates calculated from at least 3 experiments, each with 8 cultures per condition. p<0.01 for heat treatment differences and p>0.05 for heat*strain interaction by two-way ANOVA.</p