Expression，co-localization and interaction analysis of mitochondrial membrane proteins prohibitin and stomatin-like protein 2 during spermatogenesis in Larimichthys polyactis.

Hepatocellular carcinoma (HCC) treatment remains challenging due to the prevalence of metastasis and chemotherapy resistance. Mitochondrial stomatin-like protein 2 (STOML2), which is upregulated in various solid tumors, is associated with a poor prognosis; however, its biological function and molecular mechanism in HCC remain unclear. The present study aimed to elucidate the oncogenic mechanism of STOML2 in HCC and to explore its potential as a therapeutic target. Firstly, STOML2 expression in HCC and matched normal liver tissues was analyzed. In addition, STOML2-knockdown (HCCLM3-short hairpin RNA-STOML2) and -overexpression (Huh7-STOML2) cell models were established. Wound healing, Cell Counting Kit-8 and Transwell assays, and flow cytometry were performed to assess cell proliferation, invasion, migration and apoptosis in vitro. Furthermore, the biological function of STOML2 was confirmed in vivo. Co-immunoprecipitation (co-IP) and immunofluorescence staining were conducted to validate the interaction of STOML2 with prohibitin (PHB) following the prediction of binding partners. Downstream pathways regulated by STOML2 were identified using western blotting and were further investigated using the RAF1 inhibitor sorafenib. The present study revealed that STOML2 expression was significantly upregulated in HCC tissues and metastatic lesions, and was associated with poor patient prognosis. The in vitro experiments showed that STOML2 overexpression promoted proliferation, invasion, migration and autophagy, while inhibiting apoptosis in Huh7 cells. Conversely, STOML2 knockdown reversed these phenotypic changes. Furthermore, co-IP confirmed the direct interaction between STOML2 and PHB, which activated the RAF/MEK/ERK signaling pathway. The in vivo experiments further confirmed that STOML2 overexpression significantly accelerated tumor growth, whereas STOML2 or PHB knockdown inhibited tumor progression. In addition, sorafenib treatment suppressed STOML2-mediated cell migration and the expression of autophagy-related proteins by blocking the MAPK pathway. These findings elucidated the molecular mechanism by which STOML2 promotes the malignant progression of HCC and demonstrated that targeted inhibition of the PHB-MAPK pathway may reverse the pro-tumorigenic effects of STOML2. STOML2 may serve as both a prognostic biomarker and a therapeutic target in HCC. The current study provides a theoretical foundation for individualized treatment in patients with HCC and high STOML2 expression.

Results from tissue microarray in this study and our previous reports revealed that stomatin-like protein 2 (SLP-2) is notably associated with tumorigenesis and metastasis. Many members of stomatin family are involved in tumor as mitochondrial component, and recent study has revealed that SLP-2 may also function in mitochondria. To further investigate the function of SLP-2, we used siRNA target SLP-2. Data showed that knockdown of SLP-2 potently inhibited cell motility, proliferation and slightly altered cell cycle without any significant change of apoptosis. Moreover, by combined application with different chemotherapeutic reagents, we observed the enhancement of cell chemosensitivity by SLP-2 depletion. We also confirmed that, SLP-2 localizes in mitochondria, affects mitochondrial membrane potential (MMP) and ATP production. We conclude that, SLP-2 is a mitochondrial protein and therefore, functions in energy process by MMP maintenance, and subsequently affecting cell motility, proliferation and chemosensitivity.

BackgroundLoss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson’s disease (PD). We have previously identified mitochondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function.MethodsIn fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function.ResultsUsing a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations.ConclusionsThese findings place further emphasis on the importance of the protein–protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein–protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.

Asthma is an inflammatory airway disease that causes health problems worldwide. The common characteristics of asthma are airway hyperreactivity, remodeling, and airway inflammation. Thrombin and IL-8/CXCL8 play important roles in airway inflammation of asthma. Stomatin-like protein 2 (STOML2), a mitochondrial protein, is an asthma risk factor gene. In this study, we investigate whether Rho kinase (ROCK), JNK, and PEA3 participate in thrombin-induced STOML2 expression and IL-8/CXCL8 release in human lung epithelial cells. We found that thrombin induced increase in STOML2, but not prohibitin, expression in a time-dependent manner. Thrombin also induces STOML2-luciferase activity. Thrombin-induced STOML2 expression and IL-8/CXCL8 release were reduced by PEA3 siRNA. We also found that transfection of A549 cells with PEA3 siRNA reduced PEA3 protein expression. In addition, thrombin induced increase in PEA3 Ser phosphorylation and PEA3 translocation from the cytosol to the nucleus. Stimulation of cells with thrombin induced PEA3 binds to the STOML2 promoter region and PEA3-luciferase activity. SP600125 (a JNK inhibitor) and Y27632 (a ROCK inhibitor) both inhibit thrombin-caused STOML2 expression. Finally, thrombin caused JNK phosphorylation and IL-8/CXCL8 release were reduced by Y27632. Taken together, these results suggest that ROCK/JNK and PEA3 pathways play important roles in thrombin-induced STOML2 expression and IL-8/CXCL8 release in human lung epithelial cells.

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Endometriosis is a chronic gynecological disease in women of childbearing age, which leads to infertility with risk of endometrial and ovarian cancer. The pathogenesis of endometriosis is poorly understood, and cure/treatment for it is not available, except for symptomatic treatment. The recurrence rate of endometriosis is high. SLP-2 is an inner mitochondrial membrane protein whose participation has been explained in cases of endometrial stromal cell growth, differentiation and migration, but its role in endometriosis is yet to be understood. Previous studies have found altered expression of stomatin-like protein 2 (SLP-2) in the serum of endometriotic patients. Therefore, we have studied the possible role of SLP-2 in the development of endometriosis. We found the ubiquitous and high expression of SLP-2 in the endometriotic tissue of both human endometriosis patientsand rat endometriosis model. SLP-2 is seen in the glandular epithelial cells and stromal cells in the eutopic/normal or non-endometriosis group endometrium from human subjects. Finding high expression levels of SLP-2 in endometriotic tissue and ovarian cystic cells derived from endometriosis patients, we explored the possible role of SLP-2 in the cell aggregation, colonization, migration, and invasion in the human endometriotic cells associated with the progression of the endometriosis. Transient silencing of SLP-2 by its siRNA hinders endometriotic cells, aggregation, migration, and invasion into the extracellular matrix, which confirms SLP-2 involvement in endometriotic disease onset and progression. This study unravels the ubiquitous expression of SLP-2 in the human ectopic endometrial tissue and its role in the endometriotic cell migration, colonization, aggregation, and invasion leading to endometriosis progression.

During a study to characterize the proteome of lipid raft microdomains of activated human T cells we identified stomatin-like protein 2 (SLP-2). We have previously shown that T cell activation through the TCR leads to up-regulation of SLP-2 expression, which in turn correlates with enhanced T cell effector responses. Since in human T cells the major pool of SLP-2 is associated with mitochondria, we examined the function of this protein in mitochondria during T cell activation. Here, we report that up-regulation of SLP-2 expression increases mitochondrial mass and count. Such an effect correlates with an increase in the expression of the nuclear transcriptional co-activator PGC-1α, in lipid biosynthesis linked to mitochondrial membrane biogenesis, and in mitochondrial DNA replication. As expected, these changes translate into an increase in both cellular and mitochondrial ATP levels, higher mitochondrial uptake of calcium, and enhanced resistance to apoptosis through the intrinsic pathway. Based on these results we propose that SLP-2 expression is a key regulatory step for mitochondrial biogenesis and function in response to T cell activation.

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.

Mitochondria play a critical role in various pathways of regulated cell death. Here we propose a novel method for detection of initial derangement of mitochondria in degenerating and dying neuronal cells. The method is based on our recent finding that antibodies directed against the cannabinoid type 1 receptor (CB1) also bind the mitochondrial stomatin-like protein 2 (SLP2) that belongs to an inner mitochondrial membrane protein complex. It is well established that SLP2 regulates mitochondrial biogenesis and respiratory functions. We now show that anti-CB1 antibodies recognize conformational epitopes but not the linear amino acid sequence of SLP2. In addition we found that anti-CB1 serum mostly labels swollen mitochondria with early or advanced stages of pathology in mouse brain while other proteins of the complex may mask epitopes of SLP2 in the normal mitochondria. Although neurons and endothelial cells in healthy brains contain occasional immunopositive mitochondria detectable with anti-CB1 serum, their numbers increase significantly after hypoxic insults in parallel with signs of cellular damage. Moreover, use of electron microscopy suggests relocation of SLP2 from its normal functional position in the inner mitochondrial membrane into the mitochondrial matrix in pathological cells. Thus, SLP2-like immunolabeling serves as an in situ histochemical target detecting early derangement of mitochondria. Anti-CB1 serum is crucial for this purpose because available anti-SLP2 antibodies do not provide selective labeling of mitochondria in the fixed tissue. This new method of detecting mitochondrial dysfunction can benefit the in vitro research of human diseases and developmental disorders by enabling analysis in live animal models.

CHCHD10 is an amyotrophic lateral sclerosis/frontotemporal dementia gene that encodes a mitochondrial protein whose precise function is unclear. Here we show that Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing protein 10 interacts with the Stomatin-Like Protein 2 and participates in the stability of the prohibitin complex in the inner mitochondrial membrane. By using patient fibroblasts and mouse models expressing the same CHCHD10 variant (p.Ser59Leu), we show that Stomatin-Like Protein 2 forms aggregates with prohibitins, found in vivo in the hippocampus and as aggresome-like inclusions in spinal motor neurons of Chchd10S59L/+ mice. Affected cells and tissues display instability of the prohibitin complex, which participates at least in part in the activation of the OMA1 cascade with OPA1 processing leading to mitochondrial fragmentation, abnormal mitochondrial cristae morphogenesis and neuronal death found in spinal cord and the hippocampus of Chchd10S59L/+ animals. Destabilization of the prohibitin complex leads to the instability of the mitochondrial contact site and cristae organizing the system complex, probably by the disruption of OPA1-mitofilin interaction. Thus, Stomatin-Like Protein 2/prohibitin aggregates and destabilization of the prohibitin complex are critical in the sequence of events leading to motor neuron death in CHCHD10S59L-related disease.

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation.

Mitochondria are essential organelles involved in cellular energy production. The inner mitochondrial membrane protein stomatin-like protein 2 (SLP-2) is a member of the SPFH (stomatin, prohibitin, flotilin, and HflK/C) superfamily and binds to the mitochondrial glycerophospholipid cardiolipin, forming cardiolipin-enriched membrane domains to promote the assembly and/or stabilization of protein complexes involved in oxidative phosphorylation. In addition, human SLP-2 anchors a mitochondrial processing complex required for proteolytic regulation of proteins involved in mitochondrial dynamics and quality control. We now show that deletion of the gene encoding the Trypanosoma brucei homolog TbSlp2 has no effect on respiratory protein complex stability and mitochondrial functions under normal culture conditions and is dispensable for growth of T. brucei parasites. In addition, we demonstrate that TbSlp2 binds to the metalloprotease TbYme1 and together they form a large mitochondrial protein complex. The two proteins negatively regulate each other’s expression levels by accelerating protein turnover. Furthermore, we show that TbYme1 plays a role in heat-stress resistance, as TbYme1 knock-out parasites displayed mitochondrial fragmentation and loss of viability when cultured at elevated temperatures. Unbiased interaction studies uncovered putative TbYme1 substrates, some of which were differentially affected by the absence of TbYme1. Our results support emerging evidence for the presence of mitochondrial quality control pathways in this ancient eukaryote.

The FTSH4 protease is a major component of the Arabidopsis mitochondrial protein quality control system. It has both a proteolytic and a chaperone-like activity and forms complexes anchored in the inner mitochondrial membrane. Here, we show that FTSH4 assembles into two distinct forms: a dominant high-molecular-weight megacomplex with stomatin-like protein 1 (SLP1), and smaller SLP1-free assemblies. In the slp1-1 mutant, the FTSH4-SLP1 megacomplex is absent, while the abundance of SLP1-free FTSH4 assemblies is nearly doubled. Despite this, slp1-1 maintains wild-type levels of FTSH4 substrates, TIM17-2 and NAD9, indicating that the SLP1-free assemblies retain proteolytic activity. Furthermore, slp1-1 mitochondria accumulate fewer detergent-resistant HSP23.6 aggregates under elevated temperature than ftsh4-1 and even wild-type. Consequently, the mitochondrial unfolded protein response reported in ftsh4-1 is not induced in slp1-1. Although slp1-1 plants display morphological changes previously associated with ftsh4-1, such as shorter inflorescence stems due to premature arrest of the shoot apical meristem, these are less pronounced. Taken together, the increased abundance of SLP1-free FTSH4 assemblies is sufficient to support general mitochondrial proteostasis, providing effective protection against heat-induced aggregation of mitochondrial proteins. In contrast, the FTSH4-SLP1 megacomplex more effectively fulfils the meristem-specific functions of FTSH4.

In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.