Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S(2) region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S(2) LHBS mutant directly interacts with the Jun activation domain-binding protein 1 (JAB1). Association of pre-S(2) LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27(Kip1) to cytosolic proteasome for degradation. The pre-S(2) LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27(Kip1). This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S(2) mutant LHBS gene also exhibited Cdk2 activation, p27(Kip1) degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S(2) LHBS mutant causes p27(Kip1) degradation through direct interaction with JAB1. The pre-S(2) mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma.     

A novel role of proteasomal β1 subunit in tumorigenesis

p27^Kip1 is a key cell-cycle regulator whose level is primarily regulated by the ubiquitin–proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in oesophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27^Kip1directly. Interestingly, the lack of phosphorylation at Ser¹⁵⁸ of the β1 subunit promotes degradation of p27^Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27^Kip1.     

A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers

Background  

Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S₁ and -S₂ mutant large HBV surface antigen (LHBS), in which the pre-S₁ and -S₂ regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC.     

A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Layered structure of granules in upflow anaerobic sludge blanket reactor gives microbial populations resistance to metal ions

Metal ions (Cd²⁺, Cu²⁺, Ni²⁺, Zn²⁺ and Cr³⁺) did not affect glucose degradation or the production of methane during anaerobic digestion with intact and disintegrated granules from a UASB (Upflow Anaerobic Sludge Blanket) reactor. However, when Cu²⁺ was at 500 mg g^–1 VSS (volatile suspended solids) in the media, the glucose degradation rates and methane production rates decreased by 14% and 32% in disintegrated granules, respectively, whereas, in intact granules, decreases were 3% and 14%, respectively. When various electroplating metal ions were tested, 50% inhibition of acetate degradation and methane production were produced by 210–770 mg g^–1 VSS and 120–630 mg g^–1 VSS, respectively. The relative toxicity of the electroplating metals on methane production was in the order of Zn²⁺ (most toxic) > Ni²⁺ > Cu²⁺ > Cr³⁺ > Cd²⁺ (least toxic).     

The activity and selectivity of fission yeast Pop2p are affected by a high affinity for Zn2+ and Mn2+ in the active site

In eukaryotic organisms, initiation of mRNA turnover is controlled by progressive shortening of the poly-A tail, a process involving the mega-Dalton Ccr4-Not complex and its two associated 3′-5′ exonucleases, Ccr4p and Pop2p (Caf1p). RNA degradation by the 3′-5′ DEDDh exonuclease, Pop2p, is governed by the classical two metal ion mechanism traditionally assumed to be dependent on Mg²⁺ ions bound in the active site. Here, we show biochemically and structurally that fission yeast (Schizosaccharomyces pombe) Pop2p prefers Mn²⁺ and Zn²⁺ over Mg²⁺ at the concentrations of the ions found inside cells and that the identity of the ions in the active site affects the activity of the enzyme. Ion replacement experiments further suggest that mRNA deadenylation could be subtly regulated by local Zn²⁺ levels in the cell. Finally, we use site-directed mutagenesis to propose a mechanistic model for the basis of the preference for poly-A sequences exhibited by the Pop2p-type deadenylases as well as their distributive enzymatic behavior.     

Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

Extracellular Zn²⁺ activates the epithelial Na⁺ channel (ENaC) by relieving Na⁺ self-inhibition. However, a biphasic Zn²⁺ dose response was observed, suggesting that Zn²⁺ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn²⁺, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn²⁺ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn²⁺ activating site, γH239A diminished Na⁺ self-inhibition and thus concealed the activating effects of Zn²⁺. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn²⁺ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp⁵¹⁶ was predicted to be in close proximity to γHis⁸⁸. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn²⁺. Our results suggest that external Zn²⁺ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His¹⁹³, His²⁰⁰, and His²⁰² and (ii) a low-affinity Zn²⁺ inhibitory site within the palm subdomain that includes His⁸⁸ and Asp⁵¹⁶.     

Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631

Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn²⁺ binding site (His³⁰, Tyr⁵⁸, His¹³¹ and Cys¹³⁹) similar to Zn²⁺ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn²⁺ and Ca²⁺). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (T_m = 99.82°C, ΔH_cal = 4.58 × 10⁴ cal mol^-1). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.     

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values

This article utilized “protein charge ladders”—chemical derivatives of proteins with similar structure, but systematically altered net charge—to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn²⁺ stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude—by up to 7.4 units per dimer at lysosomal pH—than values predicted from standard pK_a values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn²⁺ to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn²⁺ (ΔZ < 0.44 ± 0.07 per additional Zn²⁺). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.     

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn²⁺ in this interaction because Zn²⁺ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn²⁺ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn²⁺-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn²⁺-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn²⁺ is present. These results reveal the mechanism by which Zn²⁺ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.     

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.     

Inhibition of nitrogen fixation in alfalfa by arsenate, heavy metals, fluoride, and simulated Acid rain

The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F⁻ and 42% inhibition by 100 micrograms Pb²⁺ per milliliter, to 100% inhibition by 10 micrograms Cd²⁺ per milliliter and 100 micrograms per milliliter As, Cu²⁺, and Zn²⁺ ions. Zn²⁺ showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn²⁺ > Cd²⁺ > Cu²⁺ > AsO₃⁻ > Pb²⁺ > F⁻. The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb²⁺ was applied, but was 1,000 times more sensitive to Zn²⁺. The relationship of the data to field conditions and industrial pollution is discussed.     

Cks1 regulates human hepatocellular carcinoma cell progression through osteopontin expression

Precise cell cycle regulation is critical to prevent aberrant cell proliferation and cancer progression. Cks1 was reported to be an essential accessory factor for SCF^Skp2, the ubiquitin ligase that targets p27^Kip1 for proteasomal degradation; these actions drive mammalian cell transition from G1 to S phase. In this study, we investigated the role played by Cks1 in the growth and progression of human hepatocellular carcinoma (HCC) cells. Silencing Cks1 expression abrogated osteopontin (OPN) expression in a p27^Kip1-dependent manner in Huh7 HCC cells. OPN increased the proliferation, migration and invasion of Huh7 cells. Pharmacological inhibitor studies demonstrated that ERK1/2 signaling is responsible mainly for Cks1-mediated OPN expression. Cks1 appears to regulate ERK1/2 signaling through the expression of dual-specificity phosphatase 16 (DUSP16) because both Cks1 knockdown, which leads to DUSP16 upregulation, and DUSP16 overexpression decreased ERK1/2 phosphorylation and the resulting OPN expression. The same is true for the Cks1-mediated increases in p27^Kip1, suggesting that Cks1 regulates OPN expression through activating ERK1/2 signaling either by suppressing DUSP16 expression or by a p27^Kip1-dependent mechanism. Cks1 and OPN expression levels were significantly higher, but DUSP16 expression levels were significantly lower in HCC tissues than in normal liver tissues. Both Cks1 and OPN expression were negatively correlated with DUSP16 expression, whereas Cks1 expression was positively correlated with OPN expression. Moreover, combined panels for the expression levels of Cks1, DUSP16 and OPN showed significant prognostic power for the risk assessment of HCC patient overall survival. In conclusion, our data propose a novel function for Cks1 as a tumor promoter through the expression of the strongly oncogenic protein OPN in HCC.     

AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn²⁺)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn²⁺-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn²⁺ concentrations.     

Mitotic Spindle Positioning in Saccharomyces cerevisiae Is Accomplished by Antagonistically Acting Microtubule Motor Proteins

Proper positioning of the mitotic spindle is often essential for cell division and differentiation processes. The asymmetric cell division characteristic of budding yeast, Saccharomyces cerevisiae, requires that the spindle be positioned at the mother–bud neck and oriented along the mother–bud axis. The single dynein motor encoded by the S. cerevisiae genome performs an important but nonessential spindle-positioning role. We demonstrate that kinesin-related Kip3p makes a major contribution to spindle positioning in the absence of dynein. The elimination of Kip3p function in dyn1Δ cells severely compromised spindle movement to the mother–bud neck. In dyn1Δ cells that had completed positioning, elimination of Kip3p function caused spindles to mislocalize to distal positions in mother cell bodies. We also demonstrate that the spindle-positioning defects exhibited by dyn1 kip3 cells are caused, to a large extent, by the actions of kinesin- related Kip2p. Microtubules in kip2Δ cells were shorter and more sensitive to benomyl than wild-type, in contrast to the longer and benomyl-resistant microtubules found in dyn1Δ and kip3Δ cells. Most significantly, the deletion of KIP2 greatly suppressed the spindle localization defect and slow growth exhibited by dyn1 kip3 cells. Likewise, induced expression of KIP2 caused spindles to mislocalize in cells deficient for dynein and Kip3p. Our findings indicate that Kip2p participates in normal spindle positioning but antagonizes a positioning mechanism acting in dyn1 kip3 cells. The observation that deletion of KIP2 could also suppress the inviability of dyn1Δ kar3Δ cells suggests that kinesin-related Kar3p also contributes to spindle positioning.     

Biophysical and Bioinformatic Analyses Implicate the Treponema pallidum Tp34 Lipoprotein (Tp0971) in Transition Metal Homeostasis

Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to Zn²⁺, which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions (Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺) readily induce the dimerization of Tp34; Cu²⁺ (50% effective concentration [EC₅₀] = 1.7 μM) and Zn²⁺ (EC₅₀ = 6.2 μM) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34''s likely role in metal ion homeostasis in T. pallidum.