RINGO/Speedy proteins, a family of non-canonical activators of CDK1 and CDK2.

Cyclin-dependent kinases (CDKs) require the binding to a regulatory subunit to acquire enzymatic activity, and cyclins are the canonical CDK activators. However, there are specific situations in which CDKs can be activated by non-cyclin proteins that are less characterized. This review focuses on the family of RINGO/Speedy proteins, which have no sequence amino acid homology to cyclins but can bind to and activate CDK1 and CDK2. Interestingly, RINGO/Speedy proteins can activate CDKs under conditions in which CDK-cyclin complexes would not be active, and there is evidence that RINGO/Speedy-activated CDKs can phosphorylate different sites than the cyclin-activated CDKs. RINGO/Speedy proteins were originally described in Xenopus oocytes, but their roles in mammalian cells have also been addressed. We will summarize the properties of RINGO/Speedy proteins and how they trigger CDK activation, and discuss recent studies that characterized their physiological functions. In particular, studies using genetically modified mice have shown that RingoA, also known as Spy1, plays a key role in meiosis regulation. Emerging evidence also suggests a potential role for RingoA/Spy1 in cancer.

Speedy/RINGO protein interacts with ERK/MAPK and PI3K/AKT pathways in SH-SY5Y neuroblastoma cells.

Abnormal activity of ERK/MAPK and PI3K/AKT pathways is one of the most important factors for the development of many cancer types including neuroblastoma cancer. Apart from these two pathways, some cell cycle regulators such as Speedy/RINGO also contribute to neuroblastoma development. There is data reinforcing the possible communication of the components of ERK/MAPK and PI3K/AKT pathways in carcinogenic process. In addition to this, there are studies about the direct/indirect interaction of Speedy/RINGO with these pathways in different cell types other than neuroblastoma. However, there is not any study available showing the interaction of Speedy/RINGO with both pathways in neuroblastoma cells. Therefore, the aim of this study is to determine the possible effect of Speedy/RINGO on PI3K/AKT and ERK/MAPK pathways in SH-SY5Y neuroblastoma cells. For this aim, Speedy/RINGO was silenced by siRNA technique to analyze the effects of direct inhibition of Speedy/RINGO on these pathways. Results showed that Speedy/RINGO silencing caused a significant decrease in MEK1/2 expression and AKT phosphorylation. Afterward, MEK1/2 was inhibited using a specific inhibitor U0126. Data reveal a corresponding decrease in the Speedy/RINGO expression and AKT phosphorylation indicating a reciprocal interaction between ERK/MAPK and Speedy/RINGO. In addition, MTS analysis showed that both ERK/MAPK inhibition and Speedy/RINGO silencing significantly reduced the viability of SH-SY5Y cells. This study provides information about a possible interaction of Speedy/RINGO with PI3K/AKT and ERK/MAPK pathways in SH-SY5Y cells for the first time. It will not only help to better understand the cancer-prone interactions of these pathways but also enable us to identify the appropriate molecular targets for developing efficient treatment strategies.

Translational activation of maternally derived mRNAs in oocytes and early embryos and the role of embryonic poly(A) binding protein (EPAB).

Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3'-untranslated region (3'-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process.

Exploring gene expression biomarker candidates for neurobehavioral impairment from total sleep deprivation.

BACKGROUND: Although sleep deprivation is associated with neurobehavioral impairment that may underlie significant risks to performance and safety, there is no reliable biomarker test to detect dangerous levels of impairment from sleep loss in humans. This study employs microarrays and bioinformatics analyses to explore candidate gene expression biomarkers associated with total sleep deprivation (TSD), and more specifically, the phenotype of neurobehavioral impairment from TSD. Healthy adult volunteers were recruited to a sleep laboratory for seven consecutive days (six nights). After two Baseline nights of 10 h time in bed, 11 subjects underwent an Experimental phase of 62 h of continuous wakefulness, followed by two Recovery nights of 10 h time in bed. Another six subjects underwent a well-rested Control condition of 10 h time in bed for all six nights. Blood was drawn for measuring gene expression on days two, four, and six at 4 h intervals from 08:00 to 20:00 h, corresponding to 12 timepoints across one Baseline, one Experimental, and one Recovery day. RESULTS: Altogether 212 genes changed expression in response to the TSD Treatment, with most genes exhibiting down-regulation during TSD. Also, 28 genes were associated with neurobehavioral impairment as measured by the Psychomotor Vigilance Test. The results support previous findings associating TSD with the immune response and ion signaling, and reveal novel candidate biomarkers such as the Speedy/RINGO family of cell cycle regulators. CONCLUSIONS: This study serves as an important step toward understanding gene expression changes during sleep deprivation. In addition to exploring potential biomarkers for TSD, this report presents novel candidate biomarkers associated with lapses of attention during TSD. Although further work is required for biomarker validation, analysis of these genes may aid fundamental understanding of the impact of TSD on neurobehavioral performance.

The Role of Speedy/RINGO Protein in Breast Cancer as a Future Biomarker.

Background/Aim: Cyclin-dependent kinases (CDKs) are proteins that require the binding of regulatory subunits called cyclins and play a key role in cell cycle progression and activation. CDKs play a key role in carcinogenesis of many solid malignancies, and inhibition of these proteins has produced anti-cancer effects demonstrated in preclinical studies. This narrative review was conducted to develop a hypothetical approach to determine whether Speedy/RINGO, a protein associated with CDK2, could be a possible predictive factor in breast cancer patients treated with a CDK4/6 inhibitor. Materials and Methods: A literature search was conducted in PubMed, Web of Science, Medline, and Google Scholars search engines to match the following words: "Speedy/RINGO" or "Spy1" and "CDKs" or "Cyclin-dependent kinases (CDKs)" and "CDK4/6 inhibitors" and "Regulation" and "Molecular" and "Breast cancer" and "Carcinogenesis". Only articles investigating the relationship between the Speedy/RINGO protein and CDKs at the molecular level were included. Literature information was compiled by trying to establish a relationship with our hypothesis question. Results: Speedy/RINGO is a tightly regulated proto-oncogenic mammalian protein playing important roles in the somatic cell cycle. Studies have emphasized that although it does not have amino acid sequence homology with cyclins, it can activate CDK2. In addition, results showing molecular compensation of CDK4/6 inhibition through CDK2 activation, also showed that CDK2 can predict drug resistance. Another important finding was that overexpressed Speedy/RINGO, during CDK4/6 inhibitor treatment, could strongly activate CDK2, resulting in a negative response to treatment. Conclusion: Although many predictive factors have been investigated to indicate response to CDK4/6 inhibitors or determine drug resistance, a consensus biomarker has yet to be established. In light of the information obtained from our review, it can be concluded that the Speedy/RINGO protein may have an important role as a predictive biomarker in terms of response to treatment, continuity of treatment and drug resistance in patients treated with CDK4/6 inhibitors.

Analysis and gonadal localization of Speedy A mRNA transcript, a novel gene associated with early germline cells in the scallop, Argopecten purpuratus.

The Speedy A (spdya) gene is a member of the Speedy/RINGO family, encoding a spdya protein associated with cellular cycle and meiosis in vertebrates. Results from genetic analyses indicated spdya conditional knockout mice are sterile, suggesting that this protein has essential functions in mammalian reproduction. There, however, are no published reports on the localization of spdya mRNA in the germline or in somatic cell lineages within the gonads from mollusks or other invertebrate species. Using a previously obtained transcriptome assembly from the scallop Argopecten purpuratus, an economically important hermaphroditic scallop species from Chile and Peru, there was identification of a complete coding sequence of the spdya mRNA. Phylogenetically spdya protein has sequence conservation homology with other scallops and mollusks. The relative mRNA transcript abundances at different gametogenic stages was assessed using quantitative PCR procedures. Results indicated there was an increase of spdya mRNA transcript abundance in testicular region samples at the late active stage, followed by a decrease in testis of reproductively mature individuals. To gain insight into the cellular localization of ap-spdya transcript within the gonads, specific RNA probes were synthesized for in situ hybridization analyses of gonad histological sections. Results indicated spdya mRNA is located exclusively in early germline (previtellogenic oocytes and spermatogonia) and somatic proliferative tissues of A. purpuratus ovarian and testicular regions. Overall, these results indicate there are putative functions of spdya in the early oogenesis and spermatogenesis of A. purpuratus and will contribute to furthering the understanding of gametogenesis in this species.

Salicylidene acylhydrazides attenuate survival of SH-SY5Y neuroblastoma cells through affecting mitotic regulator Speedy/RINGO and ERK/MAPK-PI3K/AKT signaling.

Salicylidene acylhydrazide group synthetic compounds ME0053, ME005 and ME0192 are known for their iron chelating properties and due to these properties they are primarily used for blocking the bacterial type 3 secretory virulence system. On the other side, targeting the metabolic pathways of iron can provide new tools for cancer prognosis and treatment. Therefore, in this study, considering their iron chelating function, the effects of the compounds ME0053, ME0055 and ME0192 were investigated in SH-SY5Y neuroblastoma cell line. Iron chelating compounds are generally known to be effective in tumor development and metastasis by targeting iron in the cell. They can exert this effect through molecules such as cyclin, CDKs, as well as signaling pathways such as PI3K/AKT and ERK/MAPK. For this reason, we analyzed the effect of the iron chelating compounds of ME0053, ME0055 and ME0192 on cell viability and proliferation rate both through ERK/MAPK and PI3K/AKT signal paths, and through the oncogenic Speedy/RINGO protein that is likely to have a regulatory effect on these two signaling pathways. Apoptosis was also investigated by measuring the amount of active caspase-3, an apoptotic marker. Along with the decrease observed in the Speedy/RINGO level, it was observed that the PI3K/AKT and ERK/MAPK signaling were decreased. This suggests that ME0053, ME0055 and ME0192 compounds significantly decrease the Speedy/RINGO expression which has a regulatory effect on the ERK/MAPK and PI3K/AKT signaling. Besides, analyzing active caspase-3 levels showed that the compounds ME0053, ME0055 and ME0192 increased its level by 218%, 60% and 175% in SH-SY5Y cells, respectively. The results of this study will pave the way for better understanding of the regulation of cancer-related ERK/MAPK and PI3K/AKT pathways and the oncogenic Speedy/RINGO which potentially affects these pathways, through synthetic salicylidene acylhydrazides and their therapeutic use in cancer.

Expression profile and potential functional differentiation of the Speedy/RINGO family in mice.

As novel cyclin-dependent kinase (CDK) activators, Speedy/RINGO (hereafter named Speedy) proteins can directly regulate the cell cycle of vertebrates by binding to and activating various CDKs. Previous studies have shown that Speedy genes are highly associated with different types of cancer and other diseases. However, Speedy genes have not been systematically identified in mice, and their function and expression profiles remain elusive, which greatly hinders the functional and mechanistic study of Speedy genes in vivo. Here, we comprehensively identified Speedy genes in the mouse genome. Phylogenetic analysis showed that the Speedy gene family should be divided into three subfamilies, rather than the previously reported two subfamilies. Mice have two of the three subfamilies of Speedy genes, namely, subfamilies A and E. Speedy subfamily C genes have been lost from the mouse genome. By combining experimental and bioinformatics approaches, we found that the genes from subfamilies A and E have different expression profiles, indicating their functional divergence, which was also consistent with the phylogenetic results. The genes belonging to subfamily E showed only slightly different expression profiles, indicating their similar functions. Coexpression network analysis showed that the genes coexpressed with mouse Speedy genes were primarily enriched in reproduction-related mechanisms and there were significant functional differences between genes from subfamilies A and E, further demonstrating functional differentiation. In summary, we provide a comprehensive landscape (from evolution to expression and function) of the Speedy family in mice; we also demonstrate that Speedy genes mainly participate in reproduction-related mechanisms and that they have undergone functional differentiation in mice.

Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation in vivo and in vitro.

Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27(Kip1). Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. In vivo neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes.

Neuroprotective strategies against calpain-mediated neurodegeneration.

Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-d-aspartate (NMDA)-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases.

The atypical cell cycle regulator Spy1 suppresses differentiation of the neuroblastoma stem cell population.

Neuroblastoma is an aggressive pediatric cancer originating embryonically from the neural crest. The heterogeneity of the disease, as most solid tumors, complicates diagnosis and treatment. In neuroblastoma this heterogeneity is well represented in both primary tumours and derived cell lines and has been shown to be driven by a population of stem-like tumour initiating cells. Resolving the molecular mediators driving the division of this population of cells may indicate effective therapeutic options for neuroblastoma patients. This study has determined that the atypical cyclin-like protein Spy1, recently indicated in driving symmetric division of glioma stem cells, is a critical factor in the stem-like properties of neuroblastoma tumor initiating cell populations. Spy1 activates Cyclin Dependent Kinases (CDK) in a manner that is unique from classical cyclins. Hence this discovery may represent an important opportunity to design CDK inhibitor drugs to uniquely target subpopulations of cells within these aggressive neural tumours.