Daniel Gaston

Organelles in Blastocystis that Blur the Distinction between Mitochondria and Hydrogenosomes

Summary Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans [1, 2]. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA [2–4]. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria [5, 6]. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes [7, 8]. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis[9]. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.

CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences

Genome engineering has been revolutionized by the discovery of clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar sequences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we developed the CRISPR MultiTargeter software. Similar DNA sequences in question are duplicated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas9 nickase targeting, as well as common and unique sgRNA target searches in 1) a set of input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. We demonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in 71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40% of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpful because it will facilitate functional genomic studies of transcript isoforms. Similarly, its application to duplicated genes may simplify multi-gene mutational targeting experiments. Overall, this program provides a unique interface that will enhance use of CRISPR/Cas technology.

Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis

Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite’s cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Evolution of the cytosolic iron-sulfur cluster assembly machinery in Blastocystis species and other microbial eukaryotes.

ABSTRACT The cytosolic iron/sulfur cluster assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages, such as animals and fungi. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. strain NandII. We identified transcripts encoding six of the components—Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18—and showed using immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation that the last three of them localized to the cytoplasm of the cell. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the last eukaryotic common ancestor (LECA). The Nbp35, Nar1 Cia1, and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic sulfur utilization factors (SUF) system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in nonmodel organisms and how it interfaces with various iron/sulfur cluster systems (i.e., the iron/sulfur cluster, nitrogen fixation, and/or SUF system) found in eukaryotic cells.

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.

Chapter 2 Predicting Proteomes of Mitochondria and Related Organelles from Genomic and Expressed Sequence Tag Data

In eukaryotes, determination of the subcellular location of a novel protein encoded in genomic or transcriptomic data provides useful clues as to its possible function. However, experimental localization studies are expensive and time-consuming. As a result, accurate in silico prediction of subcellular localization from sequence data alone is an extremely important field of study in bioinformatics. This is especially so as genomic studies expand beyond model system organisms to encompass the full diversity of eukaryotes. Here we review some of the more commonly used programs for prediction of proteins that function in mitochondria, or mitochondrion-related organelles in diverse eukaryotic lineages and provide recommendations on how to apply these methods. Furthermore, we compare the predictive performance of these programs on a mixed set of mitochondrial and non-mitochondrial proteins. Although N-terminal targeting peptide prediction programs tend to have the highest accuracy, they cannot be effectively used for partial coding sequences derived from high-throughput expressed sequence tag surveys where data for the N-terminus of the encoded protein is often missing. Therefore methods that do not rely on the presence of an N-terminal targeting sequence alone are extremely useful, especially for expressed sequence tag data. The best strategy for classification of unknown proteins is to use multiple programs, incorporating a variety of prediction strategies, and closely examine the predictions with an understanding of how each of those programs will likely handle the data.

Phenotypic overlap between familial exudative vitreoretinopathy and microcephaly, lymphedema, and chorioretinal dysplasia caused by KIF11 mutations.

IMPORTANCE Retinal detachment with avascularity of the peripheral retina, typically associated with familial exudative vitreoretinopathy (FEVR), can result from mutations in KIF11, a gene recently identified to cause microcephaly, lymphedema, and chorioretinal dysplasia (MLCRD) as well as chorioretinal dysplasia, microcephaly, and mental retardation (CDMMR). Ophthalmologists should be aware of the range of presentations for mutations in KIF11 because the phenotypic distinction between FEVR and MLCRD/CDMMR portends management implications in patients with these conditions. OBJECTIVE To identify gene mutations in patients who present with a FEVR phenotype and explore the spectrum of ocular and systemic abnormalities caused by KIF11 mutations in a cohort of patients with FEVR or microcephaly in conjunction with chorioretinopathy or FEVR. DESIGN, SETTING, AND PARTICIPANTS Clinical data and DNA were collected from each participant between 1998 and 2013 from the clinical practices of ophthalmologists and clinical geneticists internationally. Twenty-eight FEVR probands with diagnoses made by the referring physician and without a known FEVR gene mutation, and 3 with microcephaly and chorioretinopathy, were included. At least 1 patient in each pedigree manifested 1 or more of the following: macular dragging, partial retinal detachment, falciform folds, or total retinal detachment. EXPOSURES Whole-exome sequencing was conducted on affected members in multiplex pedigrees, and Sanger sequencing of the 22 exons of the KIF11 gene was performed on singletons. Clinical data and history were collected and reviewed. MAIN OUTCOMES AND MEASURES Identification of mutations in KIF11. RESULTS Four novel heterozygous KIF11 mutations and 1 previously published mutation were identified in probands with FEVR: p.A218Gfs*15, p.E470X, p.R221G, c.790-1G>T, and the previously described heterozygous p.R47X. Documentation of peripheral avascular areas on intravenous fluorescein angiography was possible in 2 probands with fibrovascular proliferation demonstrating phenotypic overlap with FEVR. CONCLUSIONS AND RELEVANCE Mutations in KIF11 cause a broader spectrum of ocular disease than previously reported, including retinal detachment. The KIF11 gene likely plays a role in retinal vascular development and mutations in this gene can lead to clinical overlap with FEVR. Cases of FEVR should be carefully inspected for the presence of microcephaly as a marker for KIF11-related disease to enhance the accuracy of the prognosis and genetic counseling.

A novel rearrangement of occludin causes brain calcification and renal dysfunction

Pediatric intracranial calcification may be caused by inherited or acquired factors. We describe the identification of a novel rearrangement in which a downstream pseudogene translocates into exon 9 of OCLN, resulting in band-like brain calcification and advanced chronic kidney disease in early childhood. SNP genotyping and read-depth variation from whole exome sequencing initially pointed to a mutation in the OCLN gene. The high degree of identity between OCLN and two pseudogenes required a combination of multiplex ligation-dependent probe amplification, PCR, and Sanger sequencing to identify the genomic rearrangement that was the underlying genetic cause of the disease. Mutations in exon 3, or at the 5–6 intron splice site, of OCLN have been reported to cause brain calcification and polymicrogyria with no evidence of extra-cranial phenotypes. Of the OCLN splice variants described, all make use of exon 9, while OCLN variants that use exons 3, 5, and 6 are tissue specific. The genetic rearrangement we identified in exon 9 provides a plausible explanation for the expanded clinical phenotype observed in our individuals. Furthermore, the lack of polymicrogyria associated with the rearrangement of OCLN in our patients extends the range of cranial defects that can be observed due to OCLN mutations.

Molecular evolution and functional divergence of X-intrinsic protein genes in plants.

X-intrinsic proteins (XIPs) are a novel class of major intrinsic proteins found in diverse organisms. Recently, XIP genes have been reported to be involved in the transport of a wide range of hydrophobic solutes; however, the evolutionary forces driving their structural and functional divergence in plants are poorly understood. In the present study, comprehensive bioinformatics analyses were performed to gain insight into the molecular and evolutionary mechanisms driving this structural and functional diversification. Phylogenetic analyses have revealed the major lineage-specific expansions of XIP genes in plants. Within the eudicots, XIP genes have diverged into Asterid and Rosid-specific phylogenetic lineages and have also undergone several independent duplications during the course of evolution. Investigation of functional divergence at the protein level showed evidence for shifting evolutionary rate and/or altered constraints on the physiochemical properties of specific amino acid sites following gene duplication. Selection pressure analyses suggest that purifying selection is the predominant evolutionary force acting on the XIP gene subfamily, along with episodic positive selection. However, only a few amino acid sites were found to be subjected to such episodic positive selection. Furthermore, protein functional divergence analysis has identified critical amino acid residues, which must be validated by future experimental studies, that could provide new insights into the role of XIPs in transport of a wide range solutes of physiological importance.