Identification of sumoylated targets in proliferating mouse spermatogonia and human testicular seminomas.

Spermatogenesis is regulated by a complex network of posttranslation modifications. Sumoylation (a modification by small ubiquitin-like modifiers, or SUMO proteins) was identified as an important cellular event in different cell types. SUMO proteins are highly expressed in the testis, and their role during spermatogenesis has begun to be elucidated. Given the important role of sumoylation in the regulation of mitosis and cancer progression in other tissues, the aim of the current study was to identify the targets of SUMO in proliferating mouse spermatogonia and human seminoma tissues and to initially examine the level of sumoylation in relation to the proliferative activity of the tissues. Using freshly purified spermatogonia and C18-4 spermatogonia cell line, mass spectrometry analysis identified several SUMO targets implicated into the proliferation of spermatogonia (such as heat shock protein 60 [HSP60] and prohibitin). Tissue array and western blot approaches showed that SUMO expression is a prominent feature of human seminomas and that the proliferative activity of the tumor tissues was positively correlated with the level of SUMO expression. Downregulation of sumoylation with si-RNA was not sufficient to significantly affect the proliferation of C18-4 spermatogonia; however, SUMO overexpression increased the proliferation rate of the cells. These data suggest that cells are more sensitive to an elevated level of SUMO, and that this situation may lead to an upregulated cellular proliferation and, possibly, cancer. Mass spectrometry analysis identified around a hundred SUMO targets in seminoma samples. Notably, many of the identified proteins (such as proliferating cell nuclear antigen [PCNA], DNA topoisomerase 2-alpha [Top2A], prohibitin, 14-3-3 protein, and others) were implicated in oncogenic transformation and cancer progression.

Role and regulation of Cdc25A phosphatase in neuron death induced by NGF deprivation or ß-amyloid.

Neuron death during development and in Alzheimer's disease (AD) is associated with aberrant regulation/induction of cell cycle proteins. However, the proximal events in this process are unknown. Cell cycle initiation requires dephosphorylation of cyclin-dependent kinases by cell division cycle 25A (Cdc25A). Here, we show that Cdc25A is essential for neuronal death in response to NGF deprivation or ß-amyloid (Aß) treatment and describe the mechanisms by which it is regulated in these paradigms. Cdc25A mRNA, protein and Cdc25A phosphatase activity were induced by NGF deprivation and Aß treatment. Enhanced Cdc25A expression was also observed in rat brains infused with Aß and in Aß-overexpressing AßPPswe-PS1dE9 mice. In cultured neurons Cdc25A inhibition by chemical inhibitors or shRNA prevented cell death and neurite degeneration caused by NGF deprivation or Aß. Additionally, Cdc25A inhibition diminished distal signaling events including Cdk-dependent elevation of phospho-pRb and subsequent caspase-3 activation. Mechanism studies revealed that Cdc25A induction by NGF deprivation and Aß is mediated by activation of Forkhead transcription factors that in turn suppress miR-21, a negative regulator of Cdc25A. Our studies thus identify Cdc25A as a required upstream element of the apoptotic cell cycle pathway that is required for neuron death in response to trophic factor deprivation and to Aß exposure and therefore as a potential target to suppress pathologic neuron death.

Parkin promotes degradation of the mitochondrial pro-apoptotic ARTS protein.

Parkinson's disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD.

Regulation of the proapoptotic ARTS protein by ubiquitin-mediated degradation.

ARTS is a mitochondrial protein that promotes apoptosis induced by a variety of proapoptotic stimulators. ARTS induces apoptosis, at least in part, through binding to and antagonizing IAPs (inhibitors of apoptosis proteins). As a result of ARTS binding to IAPs, caspase inhibition is removed and apoptosis can be executed. Here we show that high cellular levels of ARTS protein sensitize cells toward apoptosis. Accordingly, in healthy cells ARTS levels are kept low through constant ubiquitin-mediated degradation. Upon proapoptotic stimuli, the ubiquitination process is inhibited, resulting in increased levels of ARTS. Increased ARTS in turn leads to a decrease of Bcl-2 and Bcl-xL protein levels, cytochrome c release from mitochondria and apoptosis.