Xuemei Zeng

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Significance Many proteins are modified by the covalent attachment of a small polypeptide called ubiquitin to their lysine residues. Lysines in ubiquitin itself are further ubiquitinated, leading to formation of ubiquitin chains. Single ubiquitins and ubiquitin chains attached to integral membrane proteins, such as receptors, transporters, and channels, serve as molecular signals mediating endocytosis of these proteins and their subsequent targeting to lysosomes for degradation. This work uses quantitative mass spectrometry to show that activated EGF receptor is ubiquitinated by one to two short polyubiquitin chains linked via ubiquitin lysine 63 or conjugated with a single monoubiquitin. It is demonstrated that these Lys63-linked polyubiquitin chains are necessary for efficient targeting of EGF receptor to the lysosomal degradation pathway. Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.

HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β.

Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance.

Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

Mammalian heart regeneration after acute heart attacks can be induced by decellularized zebrafish cardiac extracellular matrix. Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration.

c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function

The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions.

Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes

Establishing c-Mycs (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc−/− (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc−/− livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.

A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

Objective Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells. Methodology We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1. Results and Significance We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes.

Isolation of serpin-interacting proteins in C. elegans using protein affinity purification.

Caenorhabditis elegans is a useful model organism for combining multiple imaging, genetic, and biochemical methodologies to gain more insight into the biological function of specific proteins. Combining both biochemical and genetic analyses can lead to a better understanding of how a given protein may function within the context of a network of other proteins or specific pathway. Here, we describe a protocol for the biochemical isolation of serpin-interacting proteins using affinity purification and proteomic analysis. As the knowledge of in vivo serpin interacting partners in C. elegans has largely been obtained using genetic and in vitro recombinant protein studies, this protocol serves as a complementary approach to provide insight into the biological function and regulation of serpins.

UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase.

Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B. However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with homologous to the E6-AP C terminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758–1068) results in loss of UBE3Bs ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29–58) results in loss of calmodulin binding and a significant increase in the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.

Abstract B25: Novel hepatic phenotypes caused by conditional c-Myc deletion

The transcription factor c-Myc (hereafter Myc) is among the most frequently deregulated oncoproteins. Inhibition of Myc triggers proliferative arrest of transformed cells and promotes tumor regression and/or apoptosis. Myc is also developmentally necessary and myc-/- embryos die at E9.5-10.5. However, Myc9s role in the maintenance of specific tissues has been shown to be of variable importance and necessity. For example, several studies of Myc9s role in promoting liver regeneration following partial hepatectomy (PH) have given conflicting results, although all show Myc to be generally dispensable for this function. We have used a conditional murine knockout (KO) model of Myc to study its role in liver regeneration. By employing a mycfl/fl;Alb-Cre+ model in which loss of Myc occurs perinatally, we studied non-oncogenic liver proliferation and metabolism in the absence of Myc signaling. We employed basic metabolic benchmarks of liver function including measurements of triglyceride levels, oxidative phosphorylation, and TCA cycle and electron transport chain function. At the molecular level, RNAseq was performed on isolated hepatocytes and the mitochondrial proteome was evaluated by both differential and unbiased mass spectrometry. At the time of active Myc excision, myc-/- mice had a significantly lower liver: body weight ratios relative to myc+/+ controls. However, this was reversed in older mice and was associated with the hepatic accumulation of neutral lipids, cholesterol and increased fatty acid β-oxidation in myc-/- mice. RNAseq on hepatocytes and Ingenuity Pathway analyses showed differences in 105 transcripts (q PH may provide an insufficiently sustained proliferative challenge to allow adequate evaluation of Myc9s potential role in liver regeneration. We therefore utilized a mouse model of hereditary tyrosinemia in which knockout of the gene encoding fumarylacetoacetate hydrolase (FAH) leads to hepatocellular death that can be rescued by the infusion of fah+/+ hepatocytes, which expand and eventually replace the fah-/- recipient hepatocytes. FAH-deficient animals could be rescued equally well by both myc+/+ and myc-/- hepatocytes. However, livers from the latter group showed excessive neutral lipid accumulation and fibrosis, reminiscent of non-alcoholic steatohepatitis (NASH). Taken together, our results provide unequivocal evidence that Myc is dispensable for long-term hepatic regeneration but is necessary to maintain proper lipid and steroid metabolism. In Myc9s absence the excessive accumulation of these intermediates predisposes to the development of a relatively mild pathology mimicking non-alcoholic fatty liver disease, which under the duress of chronic proliferation, progresses to a more severe NASH-like picture of end-stage liver disease. Our studies thus reveal a heretofore unappreciated role for Myc in hepatic metabolic homeostasis. Citation Format: Lia R. Edmunds, Lokendra Sharma, Peter Anthony Otero, Sonia D9Souza, James M. Dolezal, Xuemei Zeng, Ying Ding, Fei Ding, Megan E. Beck, Lisa E. Kratz, Jerry Vockley, Eric Goetzman, Donald Scott, Nathan Yates, Andrew W. Duncan, Edward V. Prochownik. Novel hepatic phenotypes caused by conditional c-Myc deletion. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B25.