Structural and functional consequences of succinate dehydrogenase subunit B mutations

Kim, E; Rath, Emma M.; Tsang, Venessa; Duff, Anthony P.; Robinson, Bruce G.; Church, W. Bret; Benn, Diane E.; Dwight, Trisha; Clifton‐Bligh, Roderick

doi:10.1530/erc-15-0099

Cited by 24 publications

(18 citation statements)

References 45 publications

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“…Alternatively, the impaired binding may arise from secondary consequences of the p.Arg46 mutation. Interestingly, our previous structural modeling of these SDHB mutations had not identified the functional impact on SDHB, as both glycine and glutamine are capable of fitting within the space left by arginine, and the electron path is not nearby [25]. The findings of our current study suggest that mutations affecting residue 46 of SDHB are pathogenic via preventing maturation of SDHB.…”

Section: Discussionmentioning

confidence: 61%

“…The SDHAF3 variant was generated in a commercially available plasmid, pCMV6-SDHAF3-Myc-DDK (RC204626, Origene); while the SDHB mutants were generated in a plasmid (pEGFP-N1; 6085–1, Clonetech) containing wild-type SDHB . Briefly, normal SDHB cDNA was generated from human adrenal total RNA and inserted into pEGFP-N1, using EcoRI and BamHI restriction sites, as previously described [25]. Sanger sequencing was used to confirm the presence of wild-type or variant sequences, and that they were in-frame with the respective tag.…”

Section: Methodsmentioning

confidence: 99%

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Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma

Dwight

Kim

et al. 2017

BMC Cancer

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BackgroundGermline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with the development of pheochromocytoma (PC) and/or paraganglioma (PGL). As assembly factors have been identified as playing a role in maturation of individual SDH subunits and assembly of the functioning SDH complex, we hypothesized that SDHAF3 variants may be associated with PC/PGL and functionality of SDH.MethodsDNA was extracted from the blood of 37 individuals (from 23 families) with germline SDH mutations and 18 PC/PGL (15 sporadic, 3 familial) and screened for mutations using a custom gene panel, containing SDHAF3 (SDH assembly factor 3) as well as eight known PC/PGL susceptibility genes. Molecular and functional consequences of an identified sequence variant of SDHAF3 were assessed in yeast and mammalian cells (HEK293).ResultsUsing massively parallel sequencing, we identified a variant in SDHAF3, c.157 T > C (p.Phe53Leu), associated with increased prevalence in familial and sporadic PC/PGL (6.6%) when compared to normal populations (1.2% [1000 Genomes], p = 0.003; 2.1% [Exome Aggregation Consortium], p = 0.0063). In silico prediction tools suggest this variant is probably damaging to protein function, hence we assessed molecular and functional consequences of the resulting amino acid change (p.Phe53Leu) in yeast and human cells. We showed that introduction of SDHAF3 p.Phe53Leu into Sdh7 (ortholog of SDHAF3 in humans) null yeast resulted in impaired function, as observed by its failure to restore SDH activity when expressed in Sdh7 null yeast relative to WT SDHAF3. As SDHAF3 is involved in maturation of SDHB, we tested the functional impact of SDHAF3 c.157 T > C and various clinically relevant SDHB mutations on this interaction. Our in vitro studies in human cells show that SDHAF3 interacts with SDHB (residues 46 and 242), with impaired interaction observed in the presence of the SDHAF3 c.157 T > C variant.ConclusionsOur studies reveal novel insights into the biogenesis of SDH, uncovering a vital interaction between SDHAF3 and SDHB. We have shown that SDHAF3 interacts directly with SDHB (residue 242 being key to this interaction), and that a variant in SDHAF3 (c.157 T > C [p.Phe53Leu]) may be more prevalent in individuals with PC/PGL, and is hypomorphic via impaired interaction with SDHB.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3486-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma

Dwight

Kim

et al. 2017

BMC Cancer

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“…indistinguishable from SDH-sufficient samples) contained SDHB mutation c.380T > G, p.Ile127Ser. This was particularly interesting to us, since we have recently described a method for measuring mutant SDH function in vitro, and this same mutation was found to have minimal impact on enzymatic function [30]. It is intriguing therefore to speculate that SDH-deficient PPGLs with normal succinate:fumarate ratios are associated with mutations that do not primarily alter SDH activity and may otherwise cause tumor development through alternative mechanisms (e.g.…”

Section: Discussionmentioning

confidence: 99%

Utility of the succinate:fumarate ratio for assessing SDH dysfunction in different tumor types

Kim

Wright

Sioson

et al. 2017

Molecular Genetics and Metabolism Reports

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ObjectiveMutations of genes encoding the four subunits of succinate dehydrogenase (SDH) have been associated with pheochromocytoma and paraganglioma (PPGLs), gastrointestinal stromal tumors (GISTs) and renal cell carcinomas (RCCs). These tumors have not been characterized in a way that reflects severity of SDH dysfunction. Mass spectrometric analysis now allows measurement of metabolites extracted from formalin fixed paraffin embedded (FFPE) specimens. We assess whether SDH deficiency in various tumor types characterized by loss of SDHB protein expression correlates with SDH dysfunction as assessed by the ratio of succinate:fumarate in FFPE specimens.Patients and methodsSections of FFPE tumor specimens from 18 PPGL, 10 GIST and 11 RCC patients with known SDHx mutation status for SDH deficiency were collected for mass spectrometric analysis of succinate and fumarate.ResultsFFPE samples showed higher succinate:fumarate ratios in SDH-deficient PPGLs compared to SDH-sufficient PPGLs. Similarly, a higher succinate:fumarate ratio was able to distinguish SDH-deficient GISTs and RCCs from their SDH-sufficient counterparts with great selectivity. Interestingly, the cut-off value of the succinate:fumarate ratio was two-folds greater in RCCs than GISTs.ConclusionAnalyzing biochemical imbalances preserved in FFPE specimens with mass spectrometry expands the method and sample type repertoire available for characterisation of multiple neoplasias associated with SDH deficiency.

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“…SDH genes, including SDHB , may be associated with a variety of tumors [15–17, 19, 23–25]. Two models have been proposed to explain how SDH loss-of-function mutations lead to tumor formation [38].…”

Section: Discussionmentioning

confidence: 99%

“…Renal tumors in patients bearing SDHB mutations present various histological subtypes [15, 19–22]. Moreover, mutations in SDHB have been implicated in neuroblastomas [13,17], thyroid carcinoma [15,19], pituitary tumors [23], gastrointestinal stromal tumors (GISTs) [24], seminoma, prostatic adenocarcinoma, and lymphoepithelial carcinoma of the stomach [25]. Reduced SDHB expression is associated with growth and de-differentiation of colorectal cancer cells [26], and shorter survival time of patients with recurrent nasopharyngeal carcinoma [27].…”

Section: Introductionmentioning

confidence: 99%

LASS5 Interacts with SDHB and Synergistically Represses p53 and p21 Activity

Jiang¹,

Li²,

Wan³

et al. 2016

CMM

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Longevity Assurance 5 (LASS5), a member of the LASS/Ceramide Synthases family, synthesizes C16-ceramide and is implicated in tumor biology. However, its precise role is not yet well understood. A yeast two-hybrid screen was performed using a human cDNA library to identify potential LASS5-interaction partners. One identified clone encodes succinate dehydrogenase subunit B (SDHB). Mammalian two-hybrid assays showed that LASS5 interacts with SDHB, and the result was also confirmed by GST pull-down and co-immunoprecipitation assays. The C-terminal fragment of SDHB was required for the interaction. LASS5 and SDHB were co-localized in COS-7 cells. LASS5 and SDHB expressions were found to be up-regulated in neuroglioma tissue. Transfection assays showed that LASS5 or SDHB expression repressed p53 or p21 reporter activity, respectively. Simultaneous LASS5 and SDHB expression resulted in stronger repression of p53 and p21 reporter activity, suggesting that LASS5 and SDHB interaction may synergistically affect transcriptional regulation of p53 and p21. Our data provide new molecular insights into potential roles of LASS5 and SDHB in tumor biology.

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Structural and functional consequences of succinate dehydrogenase subunit B mutations

Cited by 24 publications

References 45 publications

Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma

Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma

Utility of the succinate:fumarate ratio for assessing SDH dysfunction in different tumor types

LASS5 Interacts with SDHB and Synergistically Represses p53 and p21 Activity

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