Control Of Protein Stability Research Articles

The T197A Knock-in Model of Cdkn1b Gene to Study the Effects of p27 Restoration In Vivo.

The CDK inhibitor, p27kip1, encoded by the Cdkn1b gene can negatively modulate cell proliferation. The control of p27 activity during the cell cycle is regulated at multiple levels, including transcription, translation, and protein stability. The last residue of p27 (threonine 198 in human, threonine 197 in mouse) is involved in the control of protein stability. We have generated a murine knock-in model (Cdkn1b T197A) in which threonine 197 is replaced by alanine, which renders p27 protein highly unstable due to a high rate of proteasomal degradation. Expectedly, Cdkn1b T197A/T197A mice present with increased body size and weight, organomegaly, and multiple organ hyperplasia, similar to what is observed in Cdkn1b KO/KO mice. We investigated the effects exerted by the restoration of normal levels of p27 protein in the tissue of Cdkn1b T197A/T197A mice. We found that proteasome inhibition with bortezomib rescues the hyperplasia induced by the lack of p27 expression in Cdkn1b T197A/T197A but not in Cdkn1b KO/KO mice. However, BAY 11-7082, a proteasome inhibitor that stabilizes IκB but not p27, fails to rescue hyperplasia in Cdkn1b T197A/T197A mice. Bortezomib increases p27 half-life and reduces the proliferation in MEFs derived from Cdkn1b T197A/T197A but not from Cdkn1b WT/WT mice, whereas BAY 11-7082 had no effect on the protein levels of p27 and on the proliferation rate of Cdkn1b T197A/T197A MEFs.The results presented here demonstrate that Cdkn1b T197A/T197A mice represent an attractive in vivo model to investigate whether the targeting of p27 degradation machinery might prove beneficial in the treatment of a variety of human proliferative disorders caused by increased turnover of p27 protein.

STAM-binding protein regulates melanoma metastasis through SLUG stabilization

STAM-binding protein, STAMBP, is a JAMM-family deubiquitinating enzyme containing the microtubule-interacting/transport domain and STAM-binding domain. Although the biological importance of STAMBP in development has been recognized because the microcephaly-capillary malformation syndrome in human is caused by its somatic mutations, the role of STAMBP in cancer has not yet been determined. In this study, we demonstrate that STAMBP is a key molecule for regulating melanoma migration and invasion, but not survival, by knocking down STAMBP invitro. STAMBP regulates SLUG expression through a post-transcriptional mechanism to control protein stability and further contributes to the invivo metastatic potential of melanoma. Collectively, these results indicate the importance of STAMBP in melanoma metastasis by regulating SLUG. It is therefore a potential therapeutic target.

Recent progress in the investigation of ubiquitination aberrance in osteosarcoma

Ubiquitination is one of the most important post-translational modification processes in eukaryotic cells, in which the ubiquitin molecules and/or ubiquitin chains are covalently transferred to the substrate after a series of enzymatic cascade reactions involving the activating (E1), conjugating (E2) and ligating (E3) enzymes. Coupling to the 26S proteasome complex to form the Ubiquitin-Proteasome System (UPS), ubiquitination plays an essential role in controlling protein stability, thereby maintaining the dynamic balance of the key cellular proteins. Besides, ubiquitination is also involved in a wide range of protein degradation-independent events, such as gene transcription and translation, signal transduction, DNA repair and endocytosis, exerting a key function in response to exogenous stimuli and the cellular homeostasis. Similar to kinases, components of the ubiquitination system are often dysregulated, leading to a variety of diseases, such as cancer. Recently, accumulating evidence has shown an increasing number of dysregulated ubiquitination processes in osteosarcoma (OS). Herein, this review briefly provides current perspectives on the aberrance of ubiquitination-associated factors and their roles in OS, providing novel insight into potential therapeutic targets of OS.

Cell Volume Controls Protein Stability and Compactness of the Unfolded State.

Macromolecular crowding is widely accepted as one of the factors that can alter protein stability, structure, and function inside cells. Less often considered is that crowding can be dynamic: as cell volume changes, either as a result of external duress or in the course of the cell cycle, water moves in or out through membrane channels, and crowding changes in tune. Both theory and in vitro experiments predict that protein stability will be altered as a result of crowding changes. However, it is unclear how much the structural ensemble is altered as crowding changes in the cell. To test this, we look at the response of a FRET-labeled kinase to osmotically induced volume changes in live cells. We examine both the folded and unfolded states of the kinase by changing the temperature of the media surrounding the cell. Our data reveals that crowding compacts the structure of its unfolded ensemble but stabilizes the folded protein. We propose that the structure of proteins lacking a rigid, well-defined tertiary structure could be highly sensitive to both increases and decreases in cell volume. Our findings present a possible mechanism for disordered proteins to act as sensors and actuators of cell cycle or external stress events that coincide with a change in macromolecular crowding.

In-Cell Titration of Small Solutes Controls Protein Stability and Aggregation.

The components of the intracellular environment vary widely in size: from large multiprotein complexes to atomic ions. Besides water, low-molecular-weight solutes (<1 kDa) such as electrolytes, metabolites, and carbohydrates are by far the most abundant of these components. Small solutes are thus key contributors to the solvation environment in the cell. Small solutes have been known for decades to alter protein structure or activity in vitro, through their interactions with protein surfaces or hydration shells. Here we use the cell itself as our test tube, by titrating its hydration, ion, or carbohydrate composition systematically. We trigger the selective uptake of specific solutes by exposing cells to hyperosmotic media. We then measure protein structure, stability, unfolding kinetics, and aggregation in these different intracellular environments by using fast relaxation imaging. We compare these results with controls where solutes cannot enter the cell and only hydration is altered. Protein structure, thermal stability, and aggregation onset all depend on the concentration and chemical nature of the solute titrated into the cell. Our work highlights the important contributions of small solutes in defining how proteins interact within the cell and suggests that intracellular variation of the solute composition could be an important regulator of protein function.

Abstract A19: Phosphorylation results in nuclear translocation of Dicer protein that increases metabolism, accelerates aging, and alters tumorigenesis

Abstract Dicer is a well-conserved and essential gene that is required for the production of micro RNAs that play key roles during development and cancer. Several studies have associated aberrant Dicer expression in humans with developmental defects (Dicer1 syndrome) and poor prognosis in various cancers. One important mechanism of controlling protein stability and function is through post-translational modification. Recently, Extracellular signal-Regulated Kinase (ERK) dependent phosphorylation of Dicer during oogenesis has been shown to be essential in C. elegans. The phosphorylation sites in Dicer (Ser1718 and Ser1842 in mice) are well conserved and the phosphorylation is necessary and sufficient for Dicer's translocalization from cytoplasm to nucleus. We are interested in understanding the phosphorylation-mediated control of Dicer function during development and cancer. To understand the role of phospho-Dicer in mammals, we have generated five new Dicer1 alleles including phospho-mimetic mouse models with both serines replaced with aspartic acids (DicerS2D), only Ser1842 replaced with aspartic acid (DicerS1842D), and only Ser1718 replaced with aspartic acid (DicerS1718D). Phenotypic study shows that &amp;gt;75% of DicerS2D/S2D mutants and &amp;gt;60% of DicerS1842D/S1842D mutants die within five days after birth, while all heterozygous mutants and DicerS1718D/S1718D mice are phenotypically normal. The DicerS2D/S2D and DicerS1842D/S1842D survivors show signs of accelerated aging including smaller size, hunching of backbone, compromised weight gain, infertility, systemic tissue atrophy, and early lethality. We have also observed that DicerS2D/S2D animals and MEFs have increased metabolism as compared with wild type. Since aging and cancer are closely linked and altered metabolism is a hallmark of cancer, we hypothesize that phosphorylated Dicer produces an altered miRNA profile that affects metabolism, leading to developmental defects and tumorigenesis. Transcriptional profiling of MEFs and testes have shown that miRNA profiles are very different in mutants than in wild type, and the most affected pathways are metabolism and cancer. To test the role of phospho-Dicer in tumorigenesis, we crossed the DicerS2D allele into KRasLA1/+ driven nonmetastatic tumor model. The results show that phospho-Dicer reduces tumor-free survival, changes tumor spectrum, and drives metastasis in this tumor model. Citation Format: Neeraj K. Aryal, Swathi Arur, Guillermina Lozano. Phosphorylation results in nuclear translocation of Dicer protein that increases metabolism, accelerates aging, and alters tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A19.

A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria

The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device – an in vivo RBS-selector – that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17–100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria.

Two separate pathways regulate protein stability of ATM/ATR-related protein kinases Mec1 and Tel1 in budding yeast.

Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization. How TTT controls protein stability of ATM and ATR remains to be precisely determined. Here we show that Asa1, like Tel2, plays a major role in stabilization of newly synthesized Mec1 and Tel1 proteins whereas Pih1 contributes to Mec1 and Tel1 stability at high temperatures. Although Asa1 and Pih1 both interact with Tel2, no Asa1-Pih1 interaction is detected. Pih1 is distributed in both the cytoplasm and nucleus wheres Asa1 localizes largely in the cytoplasm. Asa1 and Pih1 are required for proper DNA damage checkpoint signaling. Our findings provide a model in which two different Tel2 pathways promote protein stabilization of Mec1 and Tel1 in budding yeast.

Noncovalent PEGylation through Protein-Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation.

Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein-polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-block-poly(N,N-dimethylaminoethyl) (PEG-b-PAMA) inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of α-amylase activity by PEG-b-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-b-PAMA molecule was initially located at six different placements of the x-, y-, and z-axis ±20 Å from the center of α-amylase, which showed that the PEG-b-PAMA nonspecifically bound to the α-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-b-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-b-PAMA did not interact with α-amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte.

Abstract 1403: PCNA-dependent cleavage and degradation of SDE2 regulates response to replication stress

Abstract Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function. Knowledge on such mechanism will be useful to identify novel cancer therapeutic interventions exploiting deregulated ubiquitin signaling and SDE2 activities in cancer. Citation Format: Ukhyun Jo, Winson Cai, Jingming Wang, Yoojin Kwon, Alan D. D’Andrea, Hyungjin Kim. PCNA-dependent cleavage and degradation of SDE2 regulates response to replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1403. doi:10.1158/1538-7445.AM2017-1403

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Role of Sumoylation in Alcohol Dehydrogenase 1 (ADH1) Stability and Activity in Alcoholic Liver Disease

BackgroundAlcohol abuse is a leading factor in mortality from liver disease and increases the risk for a wide range of adverse health effects. The liver, as the primary site of alcohol metabolism, is a major target of injury. The spectrum of Alcoholic Liver Diseases (ALD) includes simple steatosis, alcoholic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Alcohol dehydrogenase 1 (ADH1) is an enzyme, member of the oxidoreductase family and it is a dimeric protein in the cytosol of cells. ADH1 plays a pivotal role in contributing to the increase in rate of ethanol elimination from the blood. As the first enzyme of the major oxidative pathway of alcohol metabolism, it converts ethanol into acetaldehyde. Acetaldehyde, a major toxic ethanol metabolite, is mutagenic and carcinogenic, playing an important role in the pathogenesis of alcoholic liver disease (ALD) inducing production of reactive oxygen species (ROS) and apoptosis. SUMOylation is a post‐translational modification that modulates multiple cellular processes such as signal transduction, stress responses, cellular trafficking, protein‐protein interactions, protein‐DNA interactions, and transcriptional activity. Sumoylation is oxidative stress inducible. There are four distinct types of SUMO, small ubiquitin‐like modifier, proteins in humans (SUMO‐1, −2, 3‐ and −4). SUMOylation is often increased under oxidative stress. We recently reported that ubiquitin conjugating enzyme 9 (Ubc9), the sole E2 enzyme of SUMOylation, is induced in intragastric ethanol‐infusion (EI) treated mice. Our aim was to examine whether the dysregulated sumoylation could influence the ethanol‐induced ADH1 function in both ALD and HCC elucidating the molecular mechanism(s).MethodsMass Spectrometry by SUMO binding columns was performed to purify sumoylated proteins form total livers of Binge ethanol fed mouse model. Primary mouse hepatocytes and HepG2 cells (HCC cell line) were used to perform in vitro experiments. Protein and mRNA levels were measured using Western Blotting and RT‐PCR, respectively. Tryglicerides production was examined by Nile red staining.ResultsWe found that ADH1 is sumoylated in binge mice livers analyzing the peptide‐sequencing data from sample purified by SUMO immunoaffinity columns. In vitro experiments showed ethanol treatment increased ADH1 protein and mRNA levels in primary mouse hepatocytes and HepG2 cells. Sumoylation controls ADH1 expression and protein stability in ethanol treatment cellular model providing a protein stability in co‐treatment with ethanol. In addition, we found Ubc9 is required for ethanol‐induced triglycerides accumulation.ConclusionsWe report for the first time that ethanol‐mediated sumoylation increased ADH1 protein level in livers of binge mice and that ethanol feeding modulates the sumoylation status of the key proteins in alcoholic liver disease.. Sumoylation could play an important role in modulating ADH1 enzymatic activity by its expression and protein stability control. This novel finding thereby opens a new area of investigation examining the importance of ADH1 sumoylation in alcoholic liver disease and in HCC maybe suggesting its potential role in the therapeutic approch.Support or Funding InformationSupported by NIH grant 5 K01 AA022372‐04(Maria Lauda Tomasi)

The localization to PML nuclear bodies and stability of TRAIP/RNF206 are controlled by SUMOylation

TRAIP (TNF Receptor Associated factor Interacting Protein), also known as RNF206 (RING Finger protein 206), is an E3‐ubiquitin‐ligase protein participated in DNA damage signaling, DNA repair pathway and cell cycle progression. Post‐translational modifications of protein are important for stability control, subcellular localization and protein‐protein interaction. SUMO (Small‐ubiquitin‐like modifier) is one of the post‐translational protein modifiers and SUMOylation regulates diverse cellular processes including protein stability control, nuclear‐cytosolic transport transcriptional regulation, cell cycle progression and DNA damage response. In this study, I demonstrated that TRAIP is a new target protein of SUMOylation by western blotting, in vitro SUMOylation assay and immunofluorescence (IF). Using SUMO plot™ and SUMOylation Sites Prediction (SUMOsp) tools, I found putative SUMOylated residues of TRAIP and identified five SUMOylated sites of TRAIP using in vitro SUMOylation assay. I also discovered SUMOylation of TRAIP was crucial for localization to nucleus and PML nuclear bodies by IF. Additionally, SUMOylation of TRAIP prevented ubiquitylation and enhanced its protein stability in MG132 and cyclohexicmide (CHX) treatment experiments. To sum it up, these results demonstrates that SUMOylation regulates the subcellular localization and degradation of TRAIP. Recent studies show that TRAIP may play a role as a tumor suppressor. These findings improve the knowledge and clinical application of TRAIP for cancer therapy.Support or Funding InformationThis research was supported by Global PH.D Fellowship Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF‐2016H1A2A1909739) and by the Korean government (MSIP)(No. 2011‐0030043).

Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.

In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.

PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress.

Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.

Posttranslational Modifications and Plant-Environment Interaction.

Posttranslational modifications (PTMs) of proteins such as phosphorylation and ubiquitination are crucial for controlling protein stability, localization, and conformation. Genetic information encoded in DNA is transcribed, translated, and increases its complexity by multiple PTMs. Conformational change introduced by PTMs affects interacting partners of each proteins and their downstream signaling; therefore, PTMs are the major level of modulations of total outcome of living cells. Plants are living in harsh environment that requires unremitting physiological modulation to survive, and the plant response to various environment stresses is regulated by PTMs of proteins. This review deals with the novel knowledge of PTM-focused proteomic studies on various life conditions. PTMs are focused that mediate plant-environment interaction such as stress perception, protein homeostasis, control of energy shift, and defense by immune system. Integration of diverse signals on a protein via multiple PTMs is discussed as well, considering current situation where signal integration became an emerging area approached by systems biology into account.

From start to finish: amino-terminal protein modifications as degradation signals in plants.

Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the invivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants.

A post-translational modification cascade employing HDAC9-PIASy-RNF4 axis regulates chondrocyte hypertrophy by modulating Nkx3.2 protein stability

While Nkx3.2/Bapx1 promotes chondrogenic differentiation and plays a role in maintaining chondrocyte viability and suppressing chondrocyte hypertrophy, the regulatory mechanisms of Nkx3.2 remain poorly understood. Here we show that p300- and HDAC9-induced Nkx3.2 acetylation and de-acetylation, respectively, play critical roles in controlling Nkx3.2 protein stability. In addition, we also found that HDAC9-dependent de-acetylation of Nkx3.2 triggers PIASy-mediated sumoylation and subsequent RNF4-mediated SUMO-targeted ubiquitination. Furthermore, we demonstrate that Nkx3.2 regulation by HDAC9 can be linked to the management of chondrocyte survival and hypertrophic maturation during cartilage development. Finally, our results together reveal a novel mechanism of protein stability control involving complex interplay between acetylation, de-acetylation, sumoylation, and ubiquitination, and suggest that this post-translational modification of Nkx3.2 employing HDAC9-PIASy-RNF4 axis plays a crucial role in controlling chondrocyte viability and hypertrophic maturation during skeletal development in vertebrates.

The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor β (PDGFRβ) Internalization and Signaling

Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF β receptor (PDGFRβ) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFRβ clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFRβ, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFRβ. Phosphorylated Tyr-1021 in PDGFRβ was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLCγ. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLCγ phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFRβ are controlled by ubiquitination.

The SCF-type E3 Ubiquitin Ligases as Cancer Targets

The ubiquitin system controls protein stability and function. F-box proteins form SCF (SKP1-Cullin1-F-box protein)-type ubiquitin (E3) ligases to selectively target their substrates for degradation via the ubiquitin-proteasome pathway. Here, we review F-box proteins associated with cancer development. S-phase kinase-associated protein 2 (SKP2) (also known as FBXL1) is often overexpressed in human cancers, and functions as an oncogenic E3 ligase to degrade tumor suppressor gene products. Moreover, F-box/WD repeat-containing protein 7 (FBXW7) (also known as Fbw7) is often mutated in human cancers and functions as a tumor suppressive E3 ligase targeting oncogenic proteins for degradation. SKP2 is a potential drug target for cancer therapy and FBXW7 is useful in determining patient diagnosis, prognosis, and drug sensitivity. In this review, we also discuss other F-box proteins involved in cancer-associated cellular processes such as cell cycle control, epigenetic regulation, epithelial mesenchymal transition, apoptosis/survival, drug resistance, and DNA-damage responses.