HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation.Almost 30 yr ago, proliferating cell nuclear antigen (PCNA) was first identified in dividing cells using sera derived from patients suffering from systemic lupus erythematosus (Takasaki et al., 1981). A few years later, the “mother” of all cancer markers had been associated with DNA synthesis (Madsen and Celis, 1985), but it wasn’t until 1988 that Bauer and Burgers (1988) and Prelich and Stillman (1988) discovered that the homotrimeric clamp served as a processivity factor for DNA polymerases. In 1992, Shivji et al. (1992) showed that PCNA was required for DNA repair, and 10 yr later, it was identified as a target of ubiquitin and SUMO (small ubiquitin-like modifier) conjugation after exposure to ultraviolet light (Hoege et al., 2002). For a protein that has been in the spotlight of modern biochemistry, it is quite remarkable that almost nothing is known about its normal cellular turnover.Insight into this process comes now from the study of an unlikely regulator. In this issue, Groehler and Lannigan (2010) demonstrate that the relatively poorly characterized ERK8 (extracellular signal-regulated kinase 8) takes center stage in the regulation of PCNA stability in primary mammary epithelial cells. The ERK family of kinases belongs to the mitogen-activated protein kinase superfamily and carries a Thr-Glu-Tyr (T-E-Y) activation motif that needs to be phosphorylated to enable kinase activity (Abe et al., 2002). Interestingly, ERK8 also needs to bind to chromatin to become active. The authors identified a highly conserved PXXXP motif in the C-terminal half of ERK8 that appeared to confer autoinhibition, an activity which is relieved upon chromatin binding. Relatively close by, in the middle of ERK8, resides a PCNA-interacting peptide (PIP) box required for the interaction with PCNA (Warbrick, 1998). Curiously, only the chromatin-bound fraction of ERK8 bound to the chromatin-bound fraction of PCNA. However, a functional PIP box was not required for ERK8 to associate with nuclear DNA in the cell. These results argue that ERK8 is not anchored to chromatin by PCNA but associates with it independently. Moreover, they strongly suggest that ERK8’s PIP box binds to PCNA only when the kinase is associated with chromatin. Importantly, overexpression of an ERK8 PIP box mutant resulted in destabilization of PCNA. The effect on PCNA stability seemed to be highly specific, as depletion of ERK8 caused codepletion of PCNA but did not lead to a decrease in steady-state levels of a variety of other cell cycle regulators.Why is the interaction with PCNA confined to chromatin? The reason is likely due to the fact that ERK8’s PIP box is buried in the middle of the protein. Most PCNA-interacting proteins carry their PIP box either at the N or C terminus (Vivona and Kelman, 2003). One other well-studied example for a protein with an internal PIP box is the essential replication factor MCM10 (minichromosome maintenance protein 10). MCM10 undergoes cell cycle–regulated modification, which probably induces a conformational switch that is necessary for the PIP box–mediated interaction with PCNA (Das-Bradoo et al., 2006). In the same vein, it is conceivable that chromatin association and the accompanying relief of autoinhibition of ERK8 cause the middle portion of the kinase to change its configuration, thereby assuming a functional PIP box domain that can be recognized by PCNA. In situations in which the rapid unloading of PCNA is required, regulation of ERK8 may be the most effective way to dispose of chromatin-bound PCNA, which is known to have an exceedingly low exchange rate (Sporbert et al., 2002). Despite the fact that interaction with ERK8 is necessary to stabilize chromatin-bound PCNA, it remains unclear whether PCNA is a direct target of ERK8-mediated phosphorylation.The next goal of Groehler and Lannigan (2010) was to dissect the mechanism underlying the ERK8-regulated degradation of PCNA. Based on the consideration that physical contact between the kinase and PCNA was an integral part of the protection, they hypothesized that ERK8 might compete with an E3 ubiquitin ligase that may target PCNA via its own PIP box. This turned out to be a smart guess because the only candidate to test was the E3 ligase HDM2, the human homologue of murine double minute 2 (Momand et al., 1992). In a set of well-controlled experiments, the authors not only demonstrate that HDM2 interacts directly with and degrades PCNA when ERK8 is absent, but they also exclude indirect effects by p53 and retinoblastoma (Rb) on this process. p53 is a direct target of HDM2 and is stabilized when their interaction is inhibited (Tao and Levine, 1999). Elevated levels of p53 trigger cell cycle arrest concomitant with hypophosphorylation of Rb, but none of these changes affect the stability of PCNA. It is not hard to imagine that the loss of chromatin-bound PCNA has severe consequences for the functionality of DNA replication and repair, resulting in chromosome breakage. The authors argued that a similar level of genome instability should be visible in ERK8-depleted cells. This was indeed the case as visualized by the accumulation of γ-H2AX foci and broken DNA (Rogakou et al., 1998). Importantly, Groehler and Lannigan (2010) observed similar effects in the ERK8 PIP box mutant, further lending credence to their model. It is worthwhile pointing out that the turnover of PCNA expands the spectrum of replication factors whose degradation is tightly linked to chromatin. CDT1, a member of the prereplication complex (Cook, 2009), is rapidly degraded in the face of DNA damage. Its degradation occurs exclusively on the chromatin-associated fraction of the protein pool and is dependent on CDT1 binding to PCNA (Arias and Walter, 2005; Hu and Xiong, 2006; Senga et al., 2006).An important question that this study raises is of course to what extent, if at all, is PCNA turnover deregulated in cancer cells? The commonly high levels of PCNA in transformed cells would be most compatible with a deregulation of ERK8 and/or HDM2 to provide a significant growth advantage. Indeed, the authors show in the last part of their study that in at least two transformed cell lines, PCNA is rendered inert to the presence of ERK8. They speculate that the underlying reason is a defect in HDM2, and although this is the most likely explanation, it still needs to be validated. It will be interesting to see how common the misregulation of PCNA turnover is in cancer tissues. At this point, it is intriguing to envision a dynamic scenario in which a two-step mechanism facilitates cell transformation (Fig. 1). Initially, deregulation of ERK8 may cause PCNA levels to decrease. This would contribute to genome instability and the accumulation of new mutations, including those affecting proper function of HDM2. In step two, deregulation of HDM2 may turn things around and result in an increase of PCNA, supporting rapid proliferation.Open in a separate windowFigure 1.Role of ERK8 in maintaining genome stability. (A) In normal cells, chromatin-bound ERK8 interacts with the chromatin fraction of PCNA, which resides at the replication fork (here just shown at the leading strand for simplicity). ERK8 binding inhibits the E3 ubiquitin ligase HDM2 from interacting with PCNA. (B) In cancer cells, inactivation of ERK8 enables HDM2 to interact with and ubiquitinate PCNA, targeting it for degradation. A decrease in PCNA levels causes an increase in DNA damage, resulting in the accumulation of new mutations. These new mutations may render HDM2 nonfunctional (rectangular form), which ultimately results in an increase of PCNA stability and facilitates cell proliferation. The homotrimeric PCNA structure (Protein Data Bank ID 2OD8) was generated using the Chimera software program (Pettersen et al., 2004).     

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide,a Histone Deacetylase Inhibitor

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming.     

Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress

Relatively low or high temperatures are responsible for a variety of physiological stress responses in insects and mites. Induced thermal stress was recently associated with increased reactive oxygen species (ROS) generation, which caused oxidative damage. In this study, we examined the time-related effect of the relatively low (0, 5, 10, and 15 °C) or high (32, 35, 38, and 41 °C) temperatures on the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), and glutathione-S-transferase (GST), and the total antioxidant capacity (TEAC) of the citrus red mite, Panonychus citri (McGregor). The malondialdehyde (MDA) concentration, as a marker of lipid peroxidation in organisms, was also measured in the citrus red mite under thermal stress conditions. Results showed that SOD and GST activities were significantly increased and play an important role in the process of antioxidant response to thermal stress. Lipid peroxidation levels increased significantly (P < 0.001) and changed in a time-dependent manner. CAT and POX activity, as well as TEAC, did not vary significantly and play a minor role to remove the ROS generation. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play an important role in reducing oxidative damage in the citrus red mite.     

Characterisation of AIDS presenters and their response to antiretroviral therapy at legnano general hospital (Italy) during the period 2000-2008

The aim of this study was to characterise the AIDS presenters diagnosed between 2000 and 2008 in Legnano (Italy), and describe their initial response to highly active antiretroviral therapy (HAART) and trends over time. Seventy-six (48.7%) of 156 patients diagnosed as having AIDS in the period 2000-2008 were AIDS presenters. The proportion of AIDS presenters increased from 23.8% in 2000 to 70.6% in 2008 (p = 0.009). The major risk factors were heterosexual transmission and a foreign place of birth, and did not significantly change over time. The median CD4+ cell count at diagnosis was 30 cells/microl and the median level of HIV RNA was 5.38 log copies/ml, with no differences between the transmission risk groups. Fifteen AIDS presenters died of AIDS-defining diseases; the others started HAART (72% with 2 NRTIs + boosted PI), and 40% after a drug resistance test. The median duration of the initial HAART was 107 days. After three months, 34% of the patients had undetectable HIV-RNA levels and the median CD4+ cell count was 140 cells/microl; the corresponding figures after 12, 24 and 48 months were respectively 84%, 82.3% and 94.1%, and 310, 370 and 380 cells/microl. In conclusion, the AIDS presenters were mainly heterosexual men and immigrants. Their proportion increased significantly over time, and a substantial proportion maintained an immunovirological response to HAART.     

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2‐D PAGE with total, phospho‐ and glycoprotein staining and MALDI‐MS/MS and database searching were conducted. The results, based on fold‐change expression, revealed a down‐regulation of total protein expression by prenatal alcohol exposure in 7‐day‐old and 3‐month‐old rats. There was an up‐regulation of protein phosphorylation but a down‐regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo‐keto reductase, tumor rejection antigen gp96, fructose‐1,6‐bisphosphatase, glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, and γ‐actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S‐transferase, glucose regulated protein 58, α‐enolase, eukaryotic translation elongation factor 1 β‐2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3‐hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.     

Dexamethasone facilitates lipid accumulation and mild feed restriction improves fatty acids oxidation in skeletal muscle of broiler chicks (Gallus gallus domesticus)

Effects of dexamethasone (DEX) and mild feed restriction on the uptake and utilization of fatty acids in skeletal muscle of broiler chicks (Gallus gallus domesticus) were investigated. Male Arbor Acres chicks (7-days old, n = 30) were injected with DEX or saline for 3 days, and a feed restriction group was included. DEX enhanced circulating very low density lipoprotein (VLDL) level and the lipid accumulation in both adipose and skeletal muscle tissues. Compared with the control, liver-carnitine palmitoyltransferase 1 (L-CPT1) and AMP-activated protein kinase (AMPK) α2 mRNA level of M. biceps femoris (BF) were down-regulated significantly by DEX, while mRNA expression of lipoprotein lipase (LPL), fatty acid transport protein 1 (FATP1), heart-fatty acid binding protein (H-FABP), long-chain acyl-CoA dehydrogenase (LCAD), activities of LPL and AMPK in both skeletal muscles were not obviously affected. Feed restriction increased the mRNA expression of LPL, L-CPT1 and LCAD of M. pectoralis major (PM), and FATP1, H-FABP, L-CPT1 and LCAD of BF. In conclusion, DEX retards the growth of body mass but facilitates lipid accumulation in both adipose and skeletal muscle tissues. In contrast to the favorable effect of mild feed restriction, DEX did not alter the uptake of fatty acids in the skeletal muscle. The result suggests that DEX may promote intramyocellular lipid accumulation by suppressed fatty acid oxidation while mild feed restriction improved fatty acid oxidation in skeletal muscle, especially in red muscle. Glucocorticoids (GCs) regulated muscle fatty acid metabolism in a different way from energy deficit caused by mild feed restriction.     

Damage-specific modification of PCNA

Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork.Key words: DNA ligase I, DNA replication, Okazaki fragment processing, PCNA, ubiquitin, SUMO     

Molecular and Phylogenetic Analyses Suggest an Additional Hepatitis B Virus Genotype “I”

A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype.     

Designer Amphiphilic Short Peptides Enhance Thermal Stability of Isolated Photosystem-I

Stability of membrane protein is crucial during protein purification and crystallization as well as in the fabrication of protein-based devices. Several recent studies have examined how various surfactants can stabilize membrane proteins out of their native membrane environment. However, there is still no single surfactant that can be universally employed for all membrane proteins. Because of the lack of knowledge on the interaction between surfactants and membrane proteins, the choice of a surfactant for a specific membrane protein remains purely empirical. Here we report that a group of short amphiphilic peptides improve the thermal stability of the multi-domain protein complex photosystem-I (PS-I) in aqueous solution and that the peptide surfactants have obvious advantages over other commonly used alkyl chain based surfactants. Of all the short peptides studied, Ac-I₅K₂-CONH₂ (I₅K₂) showed the best stabilizing effect by enhancing the melting temperature of PS-I from 48.0°C to 53.0°C at concentration of 0.65 mM and extending the half life of isolated PS-I significantly. AFM experiments showed that PS-I/I₅K₂/Triton X-100 formed large and stable vesicles and thus provide interfacial environment mimicking that of native membranes, which may partly explain why I₅K₂ enhanced the thermal stability of PS-I. Hydrophobic and hydrophilic group length of I_xK_y had an important influence on the stabilization of PS-I. Our results showed that longer hydrophobic group was more effective in stabilizing PS-I. These simple short peptides therefore exhibit significant potential for applications in membrane protein studies.