In the Rat Midbrain, SG2NA and DJ-1 have Common Interactome, Including Mitochondrial Electron Transporters that are Comodulated Under Oxidative Stress.

Scaffold proteins Striatin and SG2NA assemble kinases and phosphatases into the signalling complexes called STRIPAK. Dysfunctional STRIPAKs cause cancer, cerebral cavernous malformations, etc. DJ-1, a sensor for oxidative stress, has long been associated with the Parkinson's disease, cancer, and immune disorders. SG2NA interacts with DJ-1 and Akt providing neuroprotection under oxidative stress. To dissect the role of SG2NA and DJ-1 in neuronal pathobiology, rat midbrain extracts were immunoprecipitated with SG2NA and sixty-three interacting proteins were identified. BN-PAGE followed by the LC-MS/MS showed 1030 comigrating proteins as the potential constituents of the multimeric complexes formed by SG2NA. Forty-three proteins were common between those identified by co-immunoprecipitation and the BN-PAGE. Co-immunoprecipitation with DJ-1 identified 179 interacting partners, of which forty-one also interact with SG2NA. Among those forty-one proteins immunoprecipitated with both SG2NA and DJ-1, thirty-nine comigrated with SG2NA in the BN-PAGE, and thus are bonafide constituents of the supramolecular assemblies comprising both DJ-1 and SG2NA. Among those thirty-nine proteins, seven are involved in mitochondrial oxidative phosphorylation. In rotenone-treated rats having Parkinson's like symptoms, the levels of both SG2NA and DJ-1 increased in the mitochondria; and the association of SG2NA with the electron transport complexes enhanced. In the hemi-Parkinson's model, where the rats were injected with 6-OHDA into the midbrain, the occupancy of SG2NA and DJ-1 in the mitochondrial complexes also increased. Our study thus reveals a new family of potential STRIPAK assemblies involving both SG2NA and DJ-1, with key roles in protecting midbrain from the oxidative stress.

Cellular Impacts of Striatins and the STRIPAK Complex and Their Roles in the Development and Metastasis in Clinical Cancers (Review).

Striatins (STRNs) are generally considered to be cytoplasmic proteins, with lower expression observed in the nucleus and at cell-cell contact regions. Together with protein phosphatase 2A (PP2A), STRNs form the core region of striatin-interacting phosphatase and kinase (STRIPAK) complexes through the coiled-coil region of STRN proteins, which is crucial for substrate recruitment. Over the past two decades, there has been an increasing amount of research into the biological and cellular functions of STRIPAK members. STRNs and the constituent members of the STRIPAK complex have been found to regulate several cellular functions, such as cell cycle control, cell growth, and motility. Dysregulation of these cellular events is associated with cancer development. Importantly, their roles in cancer cells and clinical cancers are becoming recognised, with several STRIPAK components found to have elevated expression in cancerous tissues compared to healthy tissues. These molecules exhibit significant diagnostic and prognostic value across different cancer types and in metastatic progression. The present review comprehensively summarises and discusses the current knowledge of STRNs and core STRIPAK members, in cancer malignancy, from both cellular and clinical perspectives.

The profile of expression of the scaffold protein SG2NA(s) differs between cancer types and its interactome in normal vis-a-vis breast tumor tissues suggests its wide roles in regulating multiple cellular pathways.

Striatin and SG2NA are scaffold proteins that form signaling complexes called STRIPAK. It has been associated with developmental abnormalities, cancer, and several other diseases. Our earlier studies have shown that SG2NA forms a complex with the cancer-associated protein DJ-1 and the signaling kinase Akt, promoting cancer cell survival. In the present study, we used bioinformatics analyses to confirm the existence of two isoforms of human SG2NA, i.e., 78 and 87 kDas. In addition, several smaller isoforms like 35 kDa were also seen in western blot analyses of human cell lysates. The expression of these isoforms varies between different cancer cell lines of human origin. Also, the protein levels do not corroborate with its transcript levels, suggesting a complex regulation of its expression. In breast tumor tissues, the expression of the 35 and 78 kDa isoforms was higher as compared to the adjacent normal tissues, while the 87 kDa isoform was found in the breast tumor tissues only. With the progression of stages of breast cancer, while the expression of 78 kDa isoform decreased, 87 kDa became undetectable. In co-immunoprecipitation assays, the profile of the SG2NA interactome in breast tumors vis-à-vis adjacent normal breast tissues showed hundreds of common proteins. Also, some proteins were interacted with SG2NA in breast tumor tissues only. We conclude that SG2NA is involved in diverse cellular pathways and has roles in cellular reprogramming during tumorigenesis of the breast.

Ectopic expression of 35 kDa and knocking down of 78 kDa SG2NAs induce cytoskeletal reorganization, alter membrane sialylation, and modulate the markers of EMT.

SG2NA is a protein of the striatin family that organizes STRIPAK complexes. It has splice variants expressing differentially in tissues. Its 78 kDa isoform regulates cell cycle, maintains homeostasis in the endoplasmic reticulum, and prevents oxidative injuries. The 35 kDa variant is devoid of the signature WD-40 repeats in the carboxy terminal, and its function is unknown. We expressed it in NIH 3T3 cells that otherwise express 78 kDa variant only. These cells (35 EE) have altered morphology, faster rate of migration, and enhanced growth as measured by the MTT assay. Similar phenotypes were also seen in cells where the endogenous 78 kDa isoform was downregulated by siRNA (78 KD). Proteomic analyses showed that several cancer-associated proteins are modulated in both 35 EE and 78 KD cells. The 35 EE cells have diffused actin fibers, distinctive ultrastructure, reduced sialylation, and increased expression of MMP2 & 9. The 78 KD cells also had diffused actin fibers and an upregulated expression of MMP2. In both cells, markers epithelial to mesenchymal transition (EMT) viz, E- & N-cadherins, ß-catenin, slug, vimentin, and ZO-1 were modulated partially in tune with the EMT process. Since NIH 3T3 cells are mesenchymal, we also expressed 35 kDa SG2NA in MCF-7 cells of epithelial origin. In these cells (MCF-7-35), the actin fibers were also diffused and the modulation of the markers was more in tune with the EMT process. However, unlike in 35 EE cells, in MCF-7-35 cells, membrane sialylation rather increased. We infer that ectopic expression of 35 kDa and downregulation of 78 kDa SG2NAs partially induce transformed phenotypes.

Subcellular dynamics of variants of SG2NA in NIH3T3 fibroblasts.

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3ß-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3ß by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3ß caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3ß by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3ß, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.

Biophysical Characterization of SG2NA Variants and their Interaction with DJ-1 and Calmodulin in vitro.

SG2NA was first discovered as nuclear autoantigen in lung and bladder cancer patient. It was named SG2NA as its expression increases during S to G2 phase of cell cycle. SG2NA/Striatin3 was classified as a member of Striatin family along with Straitin and Zinedin due to its structural and functional relatedness. At the molecular level, SG2NA is characterized by the presence of multiple protein-protein interaction domains viz., a caveolin binding motif, a coiled coil structure, Ca2+-calmodulin binding domain and a large WD-40 repeat domain in the same order from amino to the carboxyl termini. Analysis of secondary structures of 87 and 78 kDa SG2NA isoforms showed characteristic combinations of α-helix, ß-structure, ß-turns and random coil; suggesting of effective refolding after denaturation. This study for the first time establishes the structural differences between the two prevalent isoforms of SG2NA. Recently we observed that DJ-1 interacts with variants of SG2NA both in vitro and in vivo. The SG2NA isoforms purified from inclusion bodies showed the different secondary structure conformations, stability and interaction pattern for their interacting partners (DJ-1 and calmodulin) which imparts functional diversity of SG2NA. The SG2NA isoforms showed significant differential binding affinity to DJ-1 and Calmodulin.

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress.

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.

Protein phosphatase PP2A - a novel interacting partner of carnitine transporter OCTN2 (SLC22A5) in rat astrocytes.

l-Carnitine is essential for translocation of fatty acids for their mitochondrial ß-oxidation, a process shown in the brain to take place in astrocytes. Organic cation and carnitine plasma membrane transporter OCTN2 (SLC22A5) is present in astrocytes. OCTN2 activity and localization were previously shown to be regulated by protein kinase C (PKC), although no phosphorylation of the transporter was detected. In this study, mass spectrometry was used to identify rOctn2-interacting partners in astrocytes: several cytoskeletal, ribosomal, mitochondrial, heat-shock proteins, as well as proteins involved in trafficking and signaling pathways. The analysis of signaling proteins shows that Octn2 co-precipitated with PP2A phosphatase catalytical (C) and structural (A) subunits, and with its regulatory B"' subunits - striatin, SG2NA, and zinedin. The Octn2/PP2A complex is mainly detected in endoplasmic reticulum. PKC activation increases both, carnitine transport and, as shown by immunofluorescence and surface biotinylation, transporter presence in plasma membrane. It also results in phosphorylation of SG2NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2NA decreased in the complex with Octn2. PP2A inhibition with okadaic acid does not lead to Octn2 phosphorylation; however, it abolishes observed effects of PKC activation. We postulate that PKC phosphorylates SG2NA, resulting in its dissociation from the complex and transfer of Octn2 to the plasma membrane, leading to increased transporter activity. The observed interaction could affect brain functioning in vivo, both in fatty acid metabolism and in control of carnitine homeostasis, known to change in certain brain pathologies.

Anti-pseudo-PCNA type 1 (anti-SG2NA) pattern: Track down Cancer, not SLE.

OBJECTIVE: Describe the clinical significance of anti-SG2NA antibodies also called anti-pseudo-PCNA type 1 (proliferating cell nuclear antigen auto-antibodies) which are rare antinuclear antibodies (ANAs) staining distinctly S/G2 proliferative HEp-2 cells by indirect immunofluorescence. By analogy with anti-PCNA antibodies, they have been suspected to be associated with systemic lupus erythematosus (SLE), cancers or viral diseases. METHODS: From May 2006 to February 2013, 16,827 patients were tested positive for ANAs in the Laboratory of Immunology, Strasbourg, France. We retrospectively analyzed clinical and biological data from 126 patients with anti-pseudo-PCNA type 1 antibodies. RESULTS: There was a 0.75% prevalence of anti-pseudo-PCNA type 1 Abs among ANAs(+) patients. Median age was 56.9 years (standard deviation [SD] 13.4 years) with a sex ratio female/male of 1.9. Compared to ANAs(+) patients, many more patients have been hospitalized in the Oncology and Hematology Department (23% vs. 6.3%, P < 0.05). Indeed, anti-pseudo-PCNA type 1 Abs were detected in 33 patients suffering from solid and hematological cancers (26%). Another group of patients presented various auto-immune diseases but surprisingly none of our patients was affected with SLE when 5 out of 8 patients in anti-PCNAs(+) Abs group (P < 5.10(-6)) were. Finally, the presence of anti-pseudo-PCNA type 1 Abs was associated in 30 cases with other auto-Abs reflecting a more general breakdown of B cell tolerance against other self-antigens. CONCLUSION: Considering our results, explorations for tumors should be at least recommended for patients with anti-pseudo-PCNA type 1 Abs. Lupus disease is not associated with these autoAbs.

SG2NA enhances cancer cell survival by stabilizing DJ-1 and thus activating Akt.

SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth.

Tissue specific expression of SG2NA is regulated by differential splicing, RNA editing and differential polyadenylation.

SG2NA belongs to a three member Striatin subfamily of WD-40 repeat superfamily. It has multiple protein-protein interaction domains that are involved in the assembly of supra-molecular signaling complexes. Earlier we had demonstrated that there are at least five variants of SG2NA, generated by alternative splicing. We now demonstrate that a 52kDa novel variant is generated by the editing of the transcript for the 82kDa isoform. The 52kDa protein is abundant in mouse tissues but it is barely present in immortalized cells, suggesting its role in cell differentiation. Besides splicing and editing, expression of SG2NAs in tissues is also regulated by differential polyadenylation and mRNA/protein stability. Further, the longer UTR is seen only in the brain mRNA from 1month old mouse and 8-10day old chick embryo. Like alternative splicing, differential polyadenylation of Sg2na transcripts is also conserved in evolution. Taken together, these results suggest a highly versatile and dynamic mode of regulation of SG2NA with potential implications in tissue development.

STRIPAK complexes: structure, biological function, and involvement in human diseases.

The mammalian striatin family consists of three proteins, striatin, S/G2 nuclear autoantigen, and zinedin. Striatin family members have no intrinsic catalytic activity, but rather function as scaffolding proteins. Remarkably, they organize multiple diverse, large signaling complexes that participate in a variety of cellular processes. Moreover, they appear to be regulatory/targeting subunits for the major eukaryotic serine/threonine protein phosphatase 2A. In addition, striatin family members associate with germinal center kinase III kinases as well as other novel components, earning these assemblies the name striatin-interacting phosphatase and kinase (STRIPAK) complexes. Recently, there has been a great increase in functional and mechanistic studies aimed at identifying and understanding the roles of STRIPAK and STRIPAK-like complexes in cellular processes of multiple organisms. These studies have identified novel STRIPAK and STRIPAK-like complexes and have explored their roles in specific signaling pathways. Together, the results of these studies have sparked increased interest in striatin family complexes because they have revealed roles in signaling, cell cycle control, apoptosis, vesicular trafficking, Golgi assembly, cell polarity, cell migration, neural and vascular development, and cardiac function. Moreover, STRIPAK complexes have been connected to clinical conditions, including cardiac disease, diabetes, autism, and cerebral cavernous malformation. In this review, we discuss the expression, localization, and protein domain structure of striatin family members. Then we consider the diverse complexes these proteins and their homologs form in various organisms, emphasizing what is known regarding function and regulation. Finally, we explore possible roles of striatin family complexes in disease, especially cerebral cavernous malformation.

The goldfish SG2NA gene encodes two alpha-type regulatory subunits for PP-2A and displays distinct developmental expression pattern.

SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NA alpha and SG2NA beta. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the alpha-type, and are named SG2NA alpha and SG2NA alpha(+). RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.