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

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. </p

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex

<div><p>hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.</p></div

hCLE nuclear import is transcription dependent.

<p>Cultured HEK293T cells were transfected with the phCLE-PAGFP (photoactivatable GFP) plasmid and 24 h post-transfection they were treated with Actinomycin D during a 10 min pulse, washed and used for live cell microscopy. (A), Photoactivation was applied in the cytosol to visualize hCLE import. (B), Photoactivation was applied in the nucleus to visualize hCLE export. A dotted line marking the boundary of the nucleus is included in the last panels.</p

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

Identification of hCLE as monomer and dimer in denaturing gel conditions.

<p><b><i>AC</i></b><b>:</b> Accession Number of the top protein from NCBInr protein Database (non-identical NCBI protein database).</p><p><b><i>MW (Mr):</i></b> Nominal molecular weight of each protein.</p><p><b><i>pI:</i></b> Calculated pI value.</p><p><b><i>Pept (MS[MSMS]):</i></b> number of matched peptides from the top scoring protein in peptide mass fingerprinting and number of MS/MS spectra that were matched to this protein.</p><p><b><i>Score:</i></b> Mascot protein score. This number reflects the combined scores of all observed mass spectra that can be matched to amino acid sequences within that protein. A higher score indicates a more confident match.</p><p><b><i>Seq Cov:</i></b> Percentage of the database protein sequence covered by matching peptides.</p

RNA analysis.

<p>HEK293T cells were transfected with control pC-TAP or pChCLE-TAP plasmids and the RNA associated to CBD or hCLE-CBD after DNAse treatment was isolated from the purified complexes, radiolabeled and analyzed in denaturing acrylamide-urea 15% gels. (A); protein silver staining. (B); Radiolabeled RNA.</p

Transcription inhibition increases the cytosolic accumulation of hCLE, DDX1, HSPC117 and FAM98B.

<p>Cultured HEK293T cells were incubated in the absence or the presence of Actinomycin D (Act. D) during 1 h. Nuclear and cytoslic extracts were prepared and used for Western blot assays to detect the indicated proteins. (N); nuclear fraction, (C), cytosolic fraction. RNAP II and α-actin were used as markers for subcellular fractionation.</p

Transcription inhibition retains DDX1 and HSPC117 in the cytoplasm.

<p>Cultured HEK293T cells were incubated in the absence (Control) or the presence of Actynomicin D (Act. D) during 30 min, washed, fixed and processed for immunofluorescence using antibodies anti-hCLE, DDX1, HSPC117 and DAPI.</p

hCLE modulates the expression of hCLE-interacting proteins.

<p>(A); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or specific sequences for hCLE silencing (siCLE.1 and si-CLE.2) and 4 days post-infection total cell extracts were used for Western blot against the indicated proteins. (B); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or a specific sequence for hCLE silencing (siCLE.1). 5 days post-infection, the cells were left untransfected (-) or transfected (+) with a plasmid expressing a hCLE gene with 3 silent mutations that avoids its silencing (ns.hCLE). 48<b> </b>h post-transfection, nuclear and cytoplasmic fractions were prepared and used for Western blot detection of the indicated proteins.</p