Respirasome Proteins Are Regulated by Sex-Hormone Interactions in the Brain

Abstract

The existence of sex differences in disease incidence is attributed, in part, to sex differences in metabolism. Uncovering the precise mechanism driving these differences is an extraordinarily complex process influenced by genetics, endogenous hormones, sex-specific lifetime events, individual differences and external environmental/social factors. In fact, such differences may be subtle, but across a life span, increase susceptibility to a pathology. Whilst research persists in the hope of discovering an elegant biological mechanism to underpin sex differences in disease, here, we show, for the first time, that such a mechanism may be subtle in nature but influenced by multiple sex-specific factors. A proteomic dataset was generated from a gonadectomized mouse model treated with Tibolone, a menopausal hormone therapy. Following functional enrichment analysis, we identified that Alzheimer's disease and the electron transport chain-associated pathways were regulated by sex-hormone interactions. Specifically, we identified that the expression of three respirasome proteins, NDUFA2, NDUFA7 and UQCR10, is significantly altered by compounding factors that contribute to sex differences. These proteins function in bioenergetics and produce reactive oxygen species, which are each dysregulated in many diseases with sex differences in incidence. We show sex-specific reprogrammed responses to Tibolone following gonadectomy, which primarily influence the expression of proteins contributing to metabolic pathways. This further infers that metabolic differences may underpin the observed sex differences in disease, but also that hormone therapy research now has potential in exploring sex-specific interventions to produce an effective method of prevention or treatment.

Keywords: Tibolone; gonadectomy; mitochondria; proteome; respirasome; sex differences.

Figure 1

Proteins significantly regulated according to sex. Proteins with sexually dimorphic expression are presented using a volcano plot (a); the top 20 upregulated in females (b) and males (c) are shown in heatmaps. MCODE-driven PPI analysis (d) found multiple pathways to be different between males and females, including oxidative phosphorylation (purple, p = 1 × 10⁻¹¹), Parkinson’s disease (purple, p = 1.58 × 10⁻¹¹), non-alcoholic fatty liver disease (purple, p = 2 × 10⁻¹¹), axon guidance (blue, p = 1.26 × 10⁻¹⁰), nervous system development (blue, p = 1.58 × 10⁻¹⁰) and metabolism of RNA (red, p = 2.5 × 10⁻¹⁰). PPI full network enrichment analysis (e) identified organelle localisation (p = 5 × 10⁻¹¹), establishment of organelle localization (p = 1.25 × 10⁻⁹) and supramolecular fibre organization (p = 1.26 × 10⁻⁹). Functionally enriched terms were clustered and mapped into curated bubbles (f). In blue, we see the functions which are upregulated in females, and in pink, those upregulated in males. These functions are noted as potentially sexually dimorphic. Darker nodes indicate lower p values. FC: fold chance, G: gonads, nG: no gonads, C: control, T: Tibolone.

Figure 2

Regulation of proteins in the presence or absence of gonads. The impact of gonads on expression is presented using a volcano plot (a), with the top twenty upregulated proteins in the presence of gonads (b) and in their absence (c). PPI analysis with MCODE (d) identified several enriched functions, including receptor- and clathrin-mediated endocytosis (purple, p = 6.31 × 10⁻¹⁵), ribosomes (orange, p = 1 × 10⁻¹³), protein folding (red, p = 3.16 × 10⁻¹³), propanoate metabolism (blue, p = 3.16 × 10⁻⁹), UCH proteinases (green, p = 1.26 × 10⁻⁷) and membrane trafficking (yellow, p = 1.6 × 10⁻⁵). Full network analysis (e) revealed enriched functions in the regulation of protein polymerisation (p = 5 × 10⁻¹¹), the regulation of protein-containing complex assembly (p = 6.31 × 10⁻¹¹) and carbon metabolism (p = 1.6 × 10⁻¹⁰). Proteins with increased expression in the presence or absence of gonads were analysed for functional enrichment, and then, mapped together based on shared function (f). Darker nodes indicate lower p values. FC: fold chance, M: male, F: female, C: control, T: Tibolone.

Figure 3

Hormone-regulated proteins in the prefrontal cortex of mice. A single Tibolone treatment produced the greatest number of differentially expressed proteins (a). The top 20 proteins expressed in the control (b) and in Tibolone (c) are shown in heatmaps. The first MCODE-driven PPI analysis (d) revealed SRP-dependent cotranslational protein targeting to the membrane (green, p = 7.94 × 10⁻²³), Nonsense-Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) (green, p = 1 × 10⁻²²), the formation of a pool of free 40S subunits (green, p = 2.5 × 10⁻²²), mRNA splicing (orange, p = 2 × 10⁻¹⁴), the citric acid (TCA) cycle and respiratory electron transport (blue, p = 5 × 10⁻¹⁰) and clathrin-mediated endocytosis (yellow, p = 2.5 × 10⁻¹⁰) to be the most impacted functions upon Tibolone treatment. When PPI analysis was performed on all proteins in one network (e), generation of precursor metabolites and energy (p = 1.26 × 10⁻¹²) and Alzheimer’s disease (p = 5.01 × 10⁻¹²) were the most enriched functions. When all of the significantly enriched functions were mapped into clusters (f), we can see a large number of mitochondrial metabolism functions being more prevalent in the control group (blue), whilst neuronal and synapse-associated functions are more upregulated upon Tibolone treatment.

Figure 4

Two-way interaction of proteins regulated by sex–gonad interactions. (a) A total of 119 proteins were identified to be regulated by the interaction between sex and gonads. (b) PPI analysis of these proteins found Nonsense-Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) (red, p = 1.56 × 10⁻²²), respiratory electron transport (blue, 3.16 × 10⁻¹⁷), cytoplasmic ribosomal proteins (green, p = 6.3 × 10⁻¹⁴), the proteosome (orange, p = 1 × 10⁻⁸), mRNA splicing (yellow, p = 6.3 × 10⁻⁷) and post-translational protein phosphorylation (brown, p = 1.58 × 10⁻⁷) to be the top enriched functions, per cluster. PPI analysis without MCODE-driven clustering revealed Nonsense-Mediated Decay to be the most enriched function (p = 3.16 × 10⁻¹⁰) regulated by sex and gonads. (c) Functions enriched by sex–gonad interactions were also grouped by observing functions associated with RAF-MAP kinase. Of these enriched functions, the most significant GO cellular component was the cytoplasm (q = 4.14 × 10⁻¹⁷), the most significant GO molecular function was identical protein binding (q = 2.92 × 10⁻⁷), the most significant reactome pathway was metabolism (q = 1.66 × 10⁻⁵), the most significant KEGG pathway was endocrine and other factor-regulated calcium reabsorption (q = 2.63 × 10⁻⁵) and the most significant GO biological process was central nervous system development (q = 1 × 10⁻⁴).

Figure 5

Functional enrichment predicts complex 1 protein regulation to have sex-specific responses to Tibolone. (a) A total of 132 proteins were found to be regulated by sex–Tibolone interactions. (b) PPI analysis of proteins regulated by sex–Tibolone interactions identified respiratory chain complex I, mitochondria (green, p = 2.5 × 10⁻¹⁷), cytoplasmic ribosomal proteins (blue, p = 2.5 × 10⁻¹³), long-term depression (purple, p = 1 × 10⁻⁹), PIP3 activation of AKT signalling (orange, p = 1.26 × 10⁻⁸) and the citrate cycle (TCA, krebs cycle) (red, p= 3.98 × 10⁻⁷) to be the most enriched functions in each cluster. Fully connected PPI analysis found Parkinson’s disease (p = 3.98 × 10⁻¹⁰) to be the most significantly enriched function regulated by sex–Tibolone interactions. (c) When enriched functions were mapped, carbon metabolism- and autophagy-associated functions were identified to be uniquely regulated by sex–Tibolone interactions. The GO cellular component cytoplasm (q = 1.22 × 10⁻²³), KEGG pathway Alzheimer’s disease (q = 5.17 × 10⁻¹⁰), reactome pathway metabolism (q = 1.23 × 10⁻⁸), GO molecular function identical protein binding (q = 1.01 × 10⁻⁷) and GO biological process regulation of biological quality (q = 5.23 × 10⁻⁷) were the functions most enriched by sex–Tibolone interactions in each enrichment category.

Figure 6

Gonadectomy alters protein expression related to RAB geranylgeranylation, ribosomes and ATP synthesis following Tibolone treatment. (a) A total of 163 proteins were found to be regulated by gonad–Tibolone interactions. (b) PPI analysis of proteins regulated by gonad–Tibolone interactions found that RAB geranylgeranylation (green, p = 3.16 × 10⁻¹⁶), ribosomes (blue, p = 3.16 × 10⁻¹³), ATP synthesis-coupled electron transport (orange, p = 3.16 × 10⁻¹³), clathrin-mediated endocytosis (purple, p = 6.3 × 10⁻¹¹), antigen processing, ubiquitination and proteasome degradation (red, p = 2.5 × 10⁻⁹) and intermediate filament cytoskeleton organization (pink, p = 1.58x10⁻⁸) were the most enriched functions in each cluster. When PPI analysis was performed without clustering, the regulation of protein polymerisation was most significantly enriched (p = 1.25 × 10⁻⁸). (c) When enriched functions regulated by gonad–Tibolone interactions were mapped into groups, we noted that more functions associated with the immune system and endoplasmic reticulum were evident than in sex–gonad or sex–Tibolone functional enrichment analysis. The most enriched GO cellular component was the synapse (q = 1.49 × 10⁻²⁷), the most enriched GO molecular function was structural molecule activity (q = 9.36 × 10⁻¹¹), the most enriched GO biological process was regulation of cellular component organisation (q = 6 × 10⁻¹⁰), the most enriched reactome pathway was the immune system (4.95 × 10⁻⁵) and the most enriched KEGG pathway was Alzheimer’s disease (q = 0.0011) in response to gonad–Tibolone interactions.

Figure 7

Three-way interaction of proteins regulated by sex, gonads and Tibolone. A total of 139 proteins were identified to be regulated by sex–gonad–Tibolone interactions (a). PPI analysis of these proteins (b) identified respiratory electron transport (blue, p = 3.16 × 10⁻¹⁷) to be the most significant function and MCODE node cluster. This was followed by cytoplasmic ribosomal proteins (green, p = 6.3 × 10⁻¹⁴), the degradation of beta-catenin by the destruction complex (purple, p = 6.3 × 10⁻¹³), the regulation of insulin (red, p = 3.98 × 10⁻⁹) and prion diseases (orange, p = 1.58 × 10⁻⁸). When PPI analysis was performed on all proteins as a single cluster, Alzheimer’s disease was the most significant function (p = 2.5 × 10⁻¹⁰). Enriched functions were mapped into related groups (c) in which the most significant functions regulated by sex–gonad–Tibolone interactions per category were the synapse (q = 1.52 × 10⁻³⁴) in GO cellular components, transport (q = 4.68 × 10⁻⁸) in the GO biological process, Alzheimer’s disease and retrograde endocannabinoid signalling (q = 5.77 × 10⁻⁸) in KEGG pathways, metabolism (q = 2.18 × 10⁻⁵) in reactome pathways and structural molecule activity (q = 7.51 × 10⁻⁵) in GO molecular function.

Figure 8

Meta-analysis of proteins significantly regulated by sex, gonads and Tibolone. The proteins identified as being regulated by sex, gonads or Tibolone, as well as all potential interactions, were input into Metascape for functional enrichment analysis, and then, cross referenced to see which functions were significantly enriched across the most protein lists. A heatmap of the top 20 most enriched functions across each list (a) is shown, with darker squares indicating higher statistical significance. The 5 most significant functions identified were Alzheimer’s disease (q = 4.68 × 10⁻²³), metabolism of RNA (q = 1.78 × 10⁻²²), cellular response to stress (q = 9.55 × 10⁻²⁰), regulation of cellular protein localization (q = 7.24 × 10⁻¹⁹) and vesicle-mediated transport in synapse (q = 1.07 × 10⁻¹⁸). A Circos plot (b) is shown to visualize the number of shared proteins between each protein list and a network of enriched functions (c) grouped into clusters represented by the most statistically significant function in that cluster. The pie chart colours within each node correspond to the protein list in which the function is enriched, as per the colours on the Circos plot. All protein lists were then merged and a PPI analysis with MCODE clustering was performed (d); it found SRP-dependent cotranslational protein targeting to the membrane to be the most statistically enriched function overall (green, p = 1.99 × 10⁻²³), followed by oxidative phosphorylation in the red cluster (p = 1.99 × 10⁻²³), RAF activation in blue (p = 1.99 × 10⁻⁸), dicarboxylic acid metabolic process in yellow (p = 7.94 × 10⁻⁸), then, actin filament-based movement in brown, as well as aerobic respiration in yellow (p = 2.5 × 10⁻⁷) and muscle system process in purple (p = 2.5 × 10⁻⁶). When this analysis was performed without MCODE clustering (e), it revealed aerobic respiration (p = 5.01 × 10⁻¹⁴) and Alzheimer’s disease (p = 2.5 × 10⁻¹³) to be significantly enriched functions.

Figure 9

Metascape output and top regulated proteins in relation to sex, gonads and Tibolone. The top two enriched functions from the Metascape analysis of all protein lists were Alzheimer’s disease and the citric acid cycle and respiratory electron transport chain, which both belonged to the same cluster. We identified the 24 proteins shared between these functions (a) and members of the respirasome. A heatmap of the 24 respirasome proteins (b) was then produced, identifying members of complex 1, 3, 4 and 5 and cytochrome c. Only three of these proteins (c) were found to be significantly regulated by sex–gonad–Tibolone interactions: NDUFA2 (p = 0.042) and NDUFA7 (p = 0.0047) belonging to complex 1, and UQCR10 (p = 0.043), which belongs to complex 3 of the respirasome.