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- Fig. 1.
Conservation of SDHB and its mutation in heritable cancer. (A) Clustal W multiple alignment shows that C. elegans SDHB-1 has 60% identity and 84% similarity to the human homolog (identical or similar residues highlighted in blue and light blue, respectively). Three cysteine motifs characteristic of conserved iron-sulfur clusters are underlined. The boxed area and arrow indicate the critical Arg230 position. (B) Pedigree carrying mutated Arg230His SDHB, the mutation that can lead to pheochromocytoma/paraganglioma. Individual I.4 died of disseminated malignancy. Individuals I.2 (age 70) and I.3 (age 74) are tumor free; II.6 and II.7 (both 39, non-identical twins) each have tumors. (C) Genomic structure of the sdhb-1 region and gk165 deletion (dashed red lines indicate the deleted 2198 bp, including the first five exons of sdhb-1 and the first and partially the second exon of the neighboring gene, cup-15). We also analyzed a transgenic strain (see Results) containing a missense mutation G1647A in the genomic sequence (G731A in the cDNA) resulting in Arg244His.
- Fig. 2.
Phenotypic analysis of nematodes carrying either null allele gk165 or G731A missense mutation in the sdhb-1 gene. (A) Complementation of gk165 null deletion by the wild-type transgene [wild type-rescued (WTR) animals, light-blue bar] led to fertile adults in 97% of the WTR cases (n=168) relative to wild type (WT, black bar), whereas R244H worms that reached adulthood (n=83) were devoid of reproductive progeny; sdhb-1(gk165) mutants do not progress beyond L2. (B) Lifespan of deletional and point mutant animals was measured against that of the corresponding controls. sdhb-1(gk165) (n=125) mutants and R244H (n=166) transgenic animals showed a significantly shorter survival compared to their respective controls WT (n=241) and WTR (n=100) (n=3 separate experiments). (C) At 20°C, on day 3 after hatching, WT and WTR worms grew into fertile adults, while R244H animals reached adulthood only on day 4-5, suggestive of delayed development. The sdhb-1(gk165) animals showed arrested development at the L2 larval stage. (D) Wes experiment shows that the 32 kDa band specific for SDHB-1 is absent in gk165 null mutants, but is present in all control strains (WT and WTR). R244H animals also express SDHB-1 [relative to DYN-1 (93 kDa) loading control]. (E) RNA-seq data related to sdhb-1 also verify Wes results: sdhb-1 message is present in all examined strains except gk165 null mutants, for which the sdhb-1-specific mRNA lies below the detectable limit. RPKM, reads per kilobase per million mapped reads. The black horizontal line represents a threshold, below which no functional protein is observed. Statistical significance is indicated in each graph as ***P≤0.001 and **P≤0.01 versus the corresponding sample (NS, not significant; independent two-sample Student's t-test with Bonferroni correction). Error bars represent s.e.
- Fig. 3.
SDHB-1 is ubiquitously expressed throughout worm development. (A,A′) A transcriptional reporter construct shows strong expression of SDHB-1 in embryos in the comma stage (A, lateral view; A′, ventral view). (B-F) SDHB-1 expression persists throughout larval stages (B, L1; C, L2; D, L3; E, L4) into adulthood (F) with intense expression in the pharynx (D, arrowhead), together with intestinal (D, arrow) and hypodermal (E, arrowhead) cells, head and tail neurons (F, arrowheads) and the ventral nerve cord (F, arrows). In all panels, anterior is to the left and dorsal is up. These panels are composites.
- Fig. 4.
TCA cycle and related metabolites in sdhb-1 null and R244H point mutant worms and their effects on oxygen consumption. (A) TCA cycle and related metabolites: metabolites (blue) were analyzed by LC-MS in WT L2 (n=2300), L3 (n=2000), L4 (n=1750) larvae, sdhb-1(gk165) null mutants (n=2300), WTR (n=1750) and R244H point mutant (n=1750) animals. (B) Results from n=6 separate experiments measuring citrate (CIT), succinate (SUC), fumarate (FUM), malate (MAL), pyruvate (PYR), lactate (LAC) and glutamate (GLU) in deletional mutants and their developmental-stage control L2 and L3 WT animals (charts in red, dark green and light green, respectively) or in R244H mutants and their control L4 WTR and WT animals (charts in dark blue, black and light blue, respectively). Aspartate (ASP) was detected only in L4 WT and WTR animals (undetectable in L4 R244H point mutants). Alpha-ketoglutarate levels were undetectable in all (not shown). (C) The succinate-to-fumarate ratio was increased equivalently in deletional and point mutants. (D) The metabolic profile of the R244H mutant differed from that of the controls. Lactate-to-citrate and pyruvate-to-citrate ratios were calculated based on similar citrate levels in each worm type. (E,F) The effect of the deletion versus point mutation on the oxygen consumption (OCR) by Seahorse technique (n=8 parallel experiments). (E) In the case of sdhb-1(gk165) mutants (n=800), oxygen consumption was not increased after FCCP treatment compared to wild-type control. (F) R244H point mutants (n=260) failed to show accelerated oxygen consumption in response to FCCP, as also seen in the null worms. Statistical significance is indicated in each graph as ***P≤0.001 versus the corresponding sample (NS, not significant; one-way ANOVA). Error bars represent s.e.
- Fig. 5.
sdhb-1 mutants show reduced ATP production and functional mitochondrial content. (A) Confocal microscope images (40×) showing comparative mitochondrial accumulation of Mitotracker Red CMX Ros dye showing reduced fluorescence in gk165 null mutants and R244H point mutants versus respective rescue or control strains. (B) Relative fold change of the mitochondrial content in the above mutants versus stage-specific controls (n=10). (C) ATP content analyzed by ATP Bioluminescent Assay (n=2000) as relative light units (RLU). Statistical significance is indicated in each graph as ***P≤0.001, **P≤0.01 and *P≤0.05 versus the corresponding sample (independent two-sample Student's t-tests with Bonferroni correction). Error bars represent s.e.
- Fig. 6.
The effect of the Arg230His point mutation in the SDHB subunit on the structure and function of the SDHA/B enzyme complex. (A) Sequence alignment of SDHB sequences. Asp224 and Arg230, which form a salt bridge in porcine SDHB, are highly conserved, and marked in the alignment in blue and red, respectively. The positions of residues Glu154, Tyr156 and Phe226, the peptide backbone atoms of which also form hydrogen bonds with Arg230, are marked in green in the alignment. (B) Superimposition of the top-scoring wild-type (blue) and R230H (red) human SDHA/B homology models to porcine SDHA/B X-ray structure (PDB ID 4YTP; shown in white). Co-factors are shown in sphere representation, while residues 224 and 230 of SDHB are shown in stick representation for the wild-type and mutant models in blue and red, respectively. (C) Differences in averaged residue-pair contact frequencies from the 20 top-scoring models. This plot illustrates residue pairs that exhibit a loss (positive) or gain (negative) of contacts in more than 40% (>8 out of the 20 models) going from wild-type to the R230H mutant. (D) Structural annotation of the major contact changes in SDHA/B complex upon R230H mutation as seen in C (>40%). The beads in the inter-residue space indicate the location and the extent of structural perturbation. The extent of perturbation upon mutation is color coded in RWB space [R=1; W=0; B=–1], with red indicating loss of contact and blue indicating formation of a new contact. Highest perturbation is seen near the mutation site (see solid red line inset), but a significant loss of contacts is also observed at the interface of SDHA and SDHB (see dotted red line inset). FeS, iron-sulphur; FMN, flavin mononucleotide.
- Fig. 7.
Transcriptomic analysis of sdhb-1(gk165) deletional and Arg244His point mutants reveals different metabolic alterations. (A,B) Transcriptomic analysis in sdhb-1(gk165) deletional mutants (n=5000) (A) and R244H (Arg244His) point mutants (n=2500) (B), relative to their wild-type stage controls. (C) qPCR experiments performed for a subset of genes analyzed by transcriptomics in sdhb-1(gk165) deletional and R244H (Arg244His) point mutants. In A-C, dark-green and light-green columns show the ratio of mutant expression versus WT L2 (n=5000) and L3 (n=5000) controls, respectively. Black and blue columns show the ratio of mutant expression versus WT L4 (n=2500) and WTR L4 (n=2500) controls, respectively. The genes analyzed are indicated in the x-axes; fold change is represented in the y-axes. Underexpressed genes have values between 0 and 1; overexpressed genes have values from 1 upwards. Genes encoding the following enzymes were analyzed: CTS-1, citrate synthase; ACO-2, aconitase; IDH-1/2, isocitrate dehydrogenase; OGDH-1, oxoglutarate dehydrogenase; SUCA-1, succinyl-CoA ligase (ATP-specific) beta subunit; SUCG-1, succinyl-CoA ligase (GTP-specific) beta subunit; SUCL-1/2, succinyl-CoA ligase (ATP/GTP-specific) alpha subunit; SDHA-1, succinate dehydrogenase A subunit; SDHB-1, succinate dehydrogenase B subunit; MEV-1, succinate dehydrogenase C subunit; SDHD-1, succinate dehydrogenase D subunit; FUM-1, fumarase; MDH-1/2, malate dehydrogenase; ICL-1, isocitrate lyase/malate dehydrogenase; PYK-1, pyruvate kinase; PDHA-1, pyruvate dehydrogenase alpha subunit; PDHB-1, pyruvate dehydrogenase beta subunit; PCK-1/2, phosphoenolpyruvate carboxykinase; PYC-1, pyruvate carboxylase; MEN-1, malic enzyme; LDH-1, lactate dehydrogenase; GDH-1, glutamate dehydrogenase; GOT-2.2, glutamate oxoacetate transaminase.
- Fig. 8.
Arg244His point mutants exhibit a rewired metabolism compared to sdhb-1(gk165) null mutants. icl-1(RNAi) and LDH-1 inhibitor further enhance the premature developmental arrest of gk165 deletional and R244H point mutants, respectively. (A,B) A model representing metabolic changes in sdhb-1(gk165) null mutants (A) and Arg244His point mutants (B). Both mutants display elevated glyoxylate cycle activity. Only point mutants partially use their TCA cycle while exhibiting high glycolytic activity. Expression of the genes encoding enzymes labeled in blue was analyzed by qPCR. Red arrows indicate high expression; black arrows indicate normal expression. (C,D) icl-1(RNAi) treatment affected gk165 null mutants selectively: (C) embryonic lethality occurred in 42.5% of treated gk165 homozygotes (n=353), whereas gk165 animals fed HT115 bacteria expressing empty vector pPD129.36 (control RNAi) showed embryonic lethality in only 19.9% of the cases (n=315). (D) Lifespan of null mutant (n=188) and point mutant (n=155) animals was measured after ingestion of dsRNA specific for icl-1. Only null sdhb-1(gk165) animals showed a significantly shorter survival compared to their respective controls sdhb-1(gk165);control RNAi (n=148) and R244H;control RNAi (n=155) (n=3 separate experiments). (E) R244H mutants were treated by LDH-A inhibitor GSK2837808A at 1 µM and 10 µM concentrations. The compound had no effect at the lower concentration, but at 10 µM 12.4% of treated animals demonstrated increased L2/L3 larval arrest. Statistical significance is indicated in each graph as ***P≤0.001, **P≤0.01 and *P≤0.05 versus the corresponding sample (NS, not significant). Results of embryonic lethality and LDH-A inhibitor treatment were analyzed by one-way ANOVA. Lifespan measurement was analyzed by independent two-sample Student's t-test with Bonferroni correction. Error bars represent s.e.
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