Bharathi Suresh

PEST Motif Sequence Regulating Human NANOG for Proteasomal Degradation

A number of transcriptional factors are required for pluripotency of stem cells. NANOG, a homeobox transcription factor, plays a critical role in regulating embryonic stem cell (ESC) pluripotency. The expression level of NANOG is tightly regulated, and perturbation in its expression level can lead to significant difference in the morphology, expression of cell surface markers, and growth factor dependence of human and mouse ESCs. Here, we demonstrate that the proteolysis of human NANOG is regulated by the ubiquitin-proteasomal pathway. The inhibition of proteasome activity by proteasome inhibitor MG132 showed increase in protein levels of endogenous NANOG in a dose-dependent manner in human ESCs (hESCS). We demonstrated that the inhibition of the proteasome activity and cotransfection with exogenous ubiquitin promotes endogenous ubiquitination of NANOG by coimmunoprecipitation assay. In addition, we showed that both K48- and K63-branched polyubiquitin chains can conjugate with NANOG in vivo. Moreover, NANOG was an unstable protein and exhibited relatively short half-life of about 120 min in hESCs. Pretreatment of hESCs with proteasome inhibitor MG132 inhibits NANOG protein degradation and extends its half-life. Finally, we found that a PEST motif sequence (rich in proline, glutamine, serine, and threonine) from amino acid 47 to 72 located toward the N-terminus of NANOG was shown to target the protein for degradation. Deletion of the PEST motif reduced ubiquitination of NANOG, leading to NANOG stabilization. Collectively, these results indicate that the expression level, stability, and activity of NANOG are modulated by post-translational mechanisms.

The role of deubiquitinating enzymes in apoptosis

It has become apparent that ubiquitination plays a critical role in cell survival and cell death. In addition, deubiquitinating enzymes (DUBs) have been determined to be highly important regulators of these processes. Cells can be subjected to various stresses and respond in a variety of different ways ranging from activation of survival pathways to the promotion of cell death, which eventually eliminates damaged cells. The regulatory mechanisms of apoptosis depend on the balanced action between ubiquitination and deubiquitination systems. There is a growing recognition that DUBs play essential roles in regulating several binding partners to modulate the process of apoptosis. Thus, the interplay between the timing of DUB activity and the specificity of ubiquitin attachment and removal from its substrates during apoptosis is important to ensure cellular homeostasis. This review discusses the role of a few ubiquitin-specific DUBs that are involved in either promoting or suppressing the process of apoptosis.

Diverse roles of the scaffolding protein RanBPM

Ran-binding protein microtubule-organizing center (RanBPM) appears to function as a scaffolding protein in several signal transduction pathways. RanBPM is a crucial component of multiprotein complexes that regulate the cellular function by modulating and/or assembling with a wide range of proteins in different intracellular regions and thereby mediate diverse cellular functions. This suggests a role for RanBPM as a scaffolding protein. In this article, we have summarized the diverse functions of RanBPM and its interacting partners that have been investigated to date. Also, we have categorized the role of RanBPM into four divisions: RanBPM as a modulator/protein stabilizer, regulator of transcription activity, cell cycle and neurological functions.

Stability and Function of Mammalian Lethal Giant Larvae-1 Oncoprotein Are Regulated by the Scaffolding Protein RanBPM

The evolutionarily conserved lethal giant larvae (Lgl) tumor suppressor gene has an essential role in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. However, the precise molecular mechanism by which the Lgl carries out its function remains obscure. In the current study, we have identified Ran-binding protein M (RanBPM) as a novel binding partner of Mgl-1, a mammalian homolog of Drosophila tumor suppressor protein lethal (2) giant larvae (L(2)gl) by yeast two-hybrid screening. RanBPM seems to act as a scaffolding protein with a modulatory function with respect to Mgl-1. The Mgl-1 and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM resulted in inhibition of Mgl-1 degradation, and thereby extended the half-life of Mgl-1. Furthermore, the ability of Mgl-1 activity in cell migration and colony formation assay was enhanced by RanBPM. Taken together, our findings reveal that RanBPM plays a novel role in regulating Mgl-1 stability and contributes to its biological function as a tumor suppressor.

Lys-63-specific Deubiquitination of SDS3 by USP17 Regulates HDAC Activity

SDS3 is a key component of the histone deacetylase (HDAC)-dependent Sin3A co-repressor complex, serving to maintain its HDAC activity. Here, we report both exogenous and endogenous functional interaction between deubiquitinating enzyme USP17 and human SDS3 by MALDI-TOF-MS, co-immunoprecipitation assay, and GST pull-down assay. In this study, we demonstrated that SDS3 readily undergoes endogenous polyubiquitination, which is associated specifically with Lys-63-branched polyubiquitin chains and not with Lys-48-branched polyubiquitin chains. Further, we also demonstrated that USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains from SDS3 and regulates its biological functions. The deubiquitinating activity of USP17 on SDS3 negatively regulates SDS3-associated HDAC activity. The constitutive expression of USP17 and its substrate SDS3 was involved in the inhibition of anchorage-independent tumor growth and blocks cell proliferation, leading to apoptosis in cervical carcinoma cells. Furthermore, we showed that USP17 and SDS3 mutually interact with each other to regulate cancer cell viability. These data support the possibility that SDS3, being a substrate of USP17, may play an important role in developing a novel therapeutic means to inhibit specific HDAC activities in cancer.

The Importance of Ubiquitination and Deubiquitination in Cellular Reprogramming

Ubiquitination of core stem cell transcription factors can directly affect stem cell maintenance and differentiation. Ubiquitination and deubiquitination must occur in a timely and well-coordinated manner to regulate the protein turnover of several stemness related proteins, resulting in optimal embryonic stem cell maintenance and differentiation. There are two switches: an E3 ubiquitin ligase enzyme that tags ubiquitin molecules to the target proteins for proteolysis and a second enzyme, the deubiquitinating enzyme (DUBs), that performs the opposite action, thereby preventing proteolysis. In order to maintain stemness and to allow for efficient differentiation, both ubiquitination and deubiquitination molecular switches must operate properly in a balanced manner. In this review, we have summarized the importance of the ubiquitination of core stem cell transcription factors, such as Oct3/4, c-Myc, Sox2, Klf4, Nanog, and LIN28, during cellular reprogramming. Furthermore, we emphasize the role of DUBs in regulating core stem cell transcriptional factors and their function in stem cell maintenance and differentiation. We also discuss the possibility of using DUBs, along with core transcription factors, to efficiently generate induced pluripotent stem cells. Our review provides a relatively new understanding regarding the importance of ubiquitination/deubiquitination of stem cell transcription factors for efficient cellular reprogramming.

Ubiquitin-specific protease 11 functions as a tumor suppressor by modulating Mgl-1 protein to regulate cancer cell growth

The Lethal giant larvae (Lgl) gene encodes a cortical cytoskeleton protein, Lgl, and is involved in maintaining cell polarity and epithelial integrity. Previously, we observed that Mgl-1, a mammalian homologue of the Drosophila tumor suppressor protein Lgl, is subjected to degradation via ubiquitin-proteasome pathway, and scaffolding protein RanBPM prevents the turnover of the Mgl-1 protein. Consequently, overexpression of RanBPM enhances Mgl-1-mediated cell proliferation and migration. Here, we analyzed the ability of ubiquitin-specific protease 11 (USP11) as a novel regulator of Mgl-1 and it requires RanBPM to regulate proteasomal degradation of Mgl-1. USP11 showed deubiquitinating activity and stabilized Mgl-1 protein. However, USP11-mediated Mgl-1 stabilization was inhibited in RanBPM-knockdown cells. Furthermore, in the cancer cell migration, the regulation of Mgl-1 by USP11 required RanBPM expression. In addition, an in vivo study revealed that depletion of USP11 leads to tumor formation. Taken together, the results indicated that USP11 functions as a tumor suppressor through the regulation of Mgl-1 protein degradation via RanBPM.

Biological functions of hyaluronan and cytokine-inducible deubiquitinating enzymes.

The modification of proteins through post-translation and degradation by the ubiquitin-proteasome system plays a pivotal role in a broad array of biological processes. Reversal of this process by deubiquitination is a central step in the maintenance and regulation of cellular homeostasis. It now appears that the regulation of ubiquitin pathways by deubiquitinating enzymes (DUBs) could be used as targets for anticancer therapy. Recent success in inducing apoptosis in cancerous cells by USP17, a cytokine-inducible DUB encoding two hyaluronan binding motifs (HABMs) showing direct interaction with hyaluronan (HA), could prove a promising step in the development of DUBs containing HABMs as agents in anticancer therapeutics. In this review, we summarize the importance of hyaluronan (HA) in cancer, the role played by DUBs in apoptosis, and a possible relationship between DUBs and HA in cancerous cells, suggesting new strategies for applying DUB enzymes as potential anticancer therapeutics.

Hyaluronan binding motifs of USP17 and SDS3 exhibit anti-tumor activity.

Background We previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs) interacts with human SDS3 (suppressor of defective silencing 3) and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC) activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity. Methodology/Principal Findings Similarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA) using cetylpyridinium chloride (CPC) assay. Additionally, HA oligosaccharides (6-18 sugar units) competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays. Conclusions Our results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth.

K48- and K63-linked polyubiquitination of deubiquitinating enzyme USP44

Ubiquitination and deubiquitination have a critical role in protein homoeostasis in the cell. Here, we have characterized a novel USP44 (ubiquitin‐specific protease 44), which has a ZnF‐UBP (zinc‐finger ubiquitin‐specific protease) domain and conserved cysteine, histidine and asparagine/aspartic acid residues characteristic of deubiquitinating enzymes. The biochemical assay revealed that USP44 can cleave ubiquitin from ubiquitinated substrates both in vitro and in vivo. Further, USP44 undergoes both lysine 48‐ and lysine 63‐linked polyubiquitination. In situ hybridization using mouse tissues showed a basal detection level in all organs tested, with strong detection in lung, pancreas, skin, liver, stomach and intestine. RT‐PCR (reverse‐transcription PCR) analysis showed high levels of detection of USP44 mRNA in testis, spleen, lung, stomach and ovary. Furthermore, we raised a polyclonal antibody against USP44 and checked its endogenous protein expression in different cell lines. A localization study of USP44 showed its predominant expression in the nucleus.