Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Contains fulltext : 125692.pdf (publisher's version ) (Open Access)Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis

The c.214-3C>G mutation causes a severe <i>UQCC2</i> splicing defect.

<p>(A) Gel electrophoresis of full-length <i>UQCC2</i> RT-PCR products from fibroblasts grown in the absence of cycloheximide. Two prominent bands are seen in P^UQCC2 whereas only one is observed in the control. (B) Schematic diagram shows the wild-type (WT) mRNA structure and the two splice variants (1 and 2) observed in P^UQCC2. (C) Sequence chromatograms of cloned RT-PCR products show that the upper product in P^UQCC2 retains 108 bases of intronic sequence due to the use of a cryptic acceptor site, and that the lower product in P^UQCC2 lacks 14 bases of exonic sequence due to the use of an alternative cryptic acceptor site. Splice site prediction scores are from Human Splicing Finder v2.4.1 (<a href="http://www.umd.be/HSF/" target="_blank">http://www.umd.be/HSF/</a>). (D) qRT-PCR analysis using an assay that detects the exon 2/3 junction of <i>UQCC2</i> (normalized to the endogenous control <i>HPRT1</i>) demonstrates P^UQCC2 fibroblasts have only 2% wild-type <i>UQCC2</i> expression relative to controls (C¹–C⁴). P^UQCC2(1) and P^UQCC2(2) represent separate fibroblast subcultures.</p

Proposed model of CIII assembly.

<p>Complex III assembly begins with the translation activation and/or stabilization of cytochrome <i>b</i> (MTCYB) by UQCC1:UQCC2, which then delivers MTCYB to an assembly intermediate containing UQCRQ and UQCRB. This module combines with a module containing CYC1, UQCRH and UQCR10 and a module containing UQCRC2 and UQCRC1. The resulting subcomplex then dimerizes. UQCRFS1 is bound and stabilized by the CIII assembly factor LYRM7, before being incorporated into CIII with the aid of the assembly factor, BCS1L. Finally UQCR11 is added, forming the complete CIII₂. Assembly factors are indicated in gray. Proteins in which mutations are associated with complex III deficiency are bordered in red. The role of TTC19 is yet to be elucidated, although it is likely to be involved in early complex III assembly. Model adapted and updated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034-FernandezVizarra2" target="_blank">[67]</a>.</p

Depletion of the UQCC2 binding partner, UQCC1, affects complex III assembly.

<p>(A) SDS-PAGE and western blot analysis of mitochondrial extracts from HEK293 cells transfected with <i>UQCC1</i> siRNA shows lower levels of complex III subunits UQCRFS1, UQCRC1 and UQCRC2. Subunits of complex I (ND1), complex II (SDHA), complex IV (COX1) and complex V (ATP5α) are not affected by <i>UQCC1</i> knockdown. (B) BN-PAGE of HEK293 cells transfected with UQCC1 siRNA show reduced levels of holocomplex III (UQCRC2) and a mild effect on complex I in gel activity (IGA) and complex I holocomplex levels (NDUFA9). Levels of other OXPHOS complexes, complex II (SDHB), complex IV (COX2) and complex V (ATP5α) are not affected. Mock transfected cells were used as control). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s007" target="_blank">Figure S7B</a>-C for the quantification of the immunoreactive bands. (C) 2D BN-PAGE of HEK293 cells depleted of UQCC1 or cyclophilin B with indicated antibodies. The holocomplex III dimer is indicated with a line labeled CIII₂. To the right are lower molecular weight subcomplexes: UQCRC1-containing subcomplex (1) and, likely, monomeric UQCRFS1 (2). Lauryl maltoside was used to solubilize OXPHOS complexes in parts B and C. (D) Respiratory chain enzyme activity measurements of HEK293 cells transfected with <i>UQCC1</i> and <i>cyclophilin B</i> siRNAs. Mock transfected cells were set at 100%. Error bars indicate one standard deviation. Complex I ubiquinone reducing part (CI-Q), complexes II–V (CII-V) and combined activity of complex II and III (SCC) were measured relative to the activity of citrate synthase (CS).</p

UQCC2 and UQCC1 are involved in cytochrome <i>b</i> translation and/or stability.

<p>(A) SDS-PAGE analysis of ³⁵S-methionine-labeled mtDNA-encoded proteins in patient fibroblasts shows a lack of cytochrome <i>b</i> (MTCYB) protein (even at zero hours chase) suggesting a defect in cytochrome <i>b</i> synthesis or its immediate stability. (B) qRT-PCR shows normal expression of <i>cytochrome b</i> (<i>MTCYB</i>) mRNA in patient fibroblasts. (C) Autoradiogram of single step affinity purified UQCC1-TAP with ³⁵S metabolically labeled mitochondrial translation products shows UQCC1 specifically associates with newly synthesized cytochrome <i>b</i> in HEK293 cells. (D) Inhibition of mitochondrial translation in HEK293 cells results in diminished levels of UQCC1, UQCC2, mtDNA-encoded COX1, but does not affect the SDHA subunit of the nuclear encoded complex II.</p

MitoExome sequencing identifies a homozygous mutation in <i>UQCC2</i> in a patient with complex III deficiency.

<p>(A) The activity of complexes I–IV (CI-IV) as measured by spectrophotometric analysis and normalized to the activity of citrate synthase (CS), expressed as a percentage of control. Values are the average of duplicate assays. (B) Prioritization of single nucleotide variants (SNVs) and small insertion/deletions (indels) identified by MitoExome MPS. (C) Sequence chromatograms of <i>UQCC2</i> in control and patient gDNA validating the c.214-3C>G mutation detected by MitoExome sequencing.</p

Lack of UQCC2 is associated with aberrant complex III assembly, subunit expression and UQCC1 stability.

<p>(A) BN-PAGE immunoblotting of mitochondria lysed in 1% Triton X-100, using antibodies against the NDUFA9 subunit of complex I, the SDHA subunit of complex II, the UQCRC1 subunit of complex III and the COX1 subunit of complex IV shows reduced complex III assembly in P^UQCC2. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s006" target="_blank">Figure S6A</a> for quantification of immunoreactive bands. (B) SDS-PAGE and western blotting of mitochondrial lysates from patient fibroblasts demonstrate a marked deficiency of UQCC2 and UQCC1, a mild deficiency in the UQCRC2 subunit of complex III, and a more pronounced deficiency of the UQCRC1 and UQCRFS1 subunits of complex III. The P^CONTROL cell line with mutations in a complex III subunit gene showed a similar profile of complex III subunit instability but had levels of UQCC2 and UQCC1 comparable to the wild-type control. The complex II subunit SDHB and mitochondrial VDAC1 protein act as loading controls. Vertical bars indicate immunoblots performed using the same membrane. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s006" target="_blank">Figure S6A</a> for quantification of immunoreactive bands. (C) Mitochondrial lysates of HEK293 cells transfected with siRNA targeting <i>UQCC2</i> analyzed by SDS-PAGE and western blotting showed reduced levels of UQCC2 and UQCC1 proteins. As control, <i>cyclophilin B</i> knockdown and mock transfected cells were used. The asterisk indicates a non-specific, cross-reactive species. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s007" target="_blank">Figure S7A</a> for quantification of immunoreactive bands.</p

UQCC2 interacts with mitochondrial protein UQCC1.

<p>(A) SDS-PAGE analysis of HEK293 cellular fractions shows that UQCC1 is enriched in the mitochondrial fraction, similar to the mitochondrial protein TOM20. A cytosolic marker creatine kinase B-type (CK-B) was used. TC: Total Cell, Cyt: Cytoplasmic fraction, Mit: Mitochondrial fraction. (B) Proteinase K protection assay performed using mitochondria with digitonin-permeabilized outer membranes shows localization of UQCC1 within the mitochondrial inner membrane. UQCC1, unlike outer membrane localized TOM20 and the inter-membrane localized part of OXA1L, is protected from proteolysis and degraded only after the inner membrane is dissolved with Triton X-100. Western blot analysis of single step affinity purified (C) UQCC2- and (D) UQCC1-TAP from doxycycline-induced HEK293 cells shows that UQCC1 co-purifies with UQCC2-TAP and UQCC2 co-purifies with UQCC1-TAP. Additional probing of the membranes for the complex III structural subunits UQCRC1, UQCRC2, UQCRFS1 and mitochondrial ribosomal subunits MRPS22 and MRPL12 did not reveal co-elution of these proteins. Asterisks with these subunits, including the one with UQCRFS1, correspond to bands at different heights that result from previous incubations. Complex II subunit SDHA was used to rule out non-specific protein binding. Non-induced cells were used as control. Antibodies used are indicated at the left. Arrowheads indicate endogenous UQCC1 and UQCC2.</p