TRAF2 regulates the protein stability of HIPK2.

A nuclear serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of development and DNA damage response. HIPK2 can induce apoptosis under cellular stress conditions and thus its protein level is maintained low by constant proteasomal degradation. In the present study, we present evidence that TNF receptor-associated factor 2 (TRAF2) regulates the protein stability of HIPK2. Overexpression of TRAF2 decreased while its knockdown increased the HIPK2 protein level. The TRAF2-mediated decrease in HIPK2 protein expression was blocked by proteasomal inhibitor. In addition, TRAF2 decreased the protein half-life of HIPK2. We found that HIPK2 and TRAF2 co-immunoprecipitated. Interestingly, the co-immunoprecipitation was reduced while HIPK2 protein level increased following TNFα treatment, suggesting TNFα induced dissociation of TRAF2 from HIPK2 to accumulate HIPK2. Inhibition of HIPK2 partially suppressed TNFα-induced cell death, indicating that the accumulated HIPK2 may contribute to the TNFα-induced cell death. Our results suggest that TRAF2 can regulate proapoptotic function of HIPK2 by promoting proteasomal degradation.

Antioxidant Responses are Crucial for Defense against Misfolded Human Z-Type α1-Antitrypsin.

BACKGROUND: The Z-type variant of human α1-antitrypsin is involved in liver cirrhosis and pulmonary emphysema. Due to its slow folding characteristics, this variant accumulates folding intermediates and forms protein aggregates within hepatocytes. Misfolded proteins may induce oxidative stress and subsequent cell death. OBJECTIVE: The potential application of antioxidant response signaling pathway and antioxidants to cope with Z-type α1-antitrypsin-induced oxidative stress was evaluated. METHODS: Overexpression of Z-type α1-antitrypsin in Saccharomyces cerevisiae provoked oxidative stress and increased susceptibility to oxidative challenges such as hydrogen peroxide treatment. Deletion of antioxidant-response genes, including yap1, skn7, sod2, tsa1, and pst2, exacerbated the slow growth phenotype of Z-type α1-antitrypsin-expressing cells. Antioxidant treatment alleviated oxidative stress and cytotoxicity induced by Z-type α1-antitrypsin. RESULTS: Our results show that cellular antioxidant capacity is crucial to protection against misfolded Z-type α1-antitrypsin. CONCLUSION: The information obtained here may be used to prevent oxidative stress caused by misfolded proteins, which are associated with several degenerative diseases, including amyotrophic lateral sclerosis and Parkinson's disease.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance

ABSTRACT Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

An intrinsically disordered domain in Polaribacter irgensii KOPRI 22228 CspB confers extraordinary freeze-tolerance.

Organisms living in extremely cold environments possess mechanisms to survive low temperatures. Among the known cold-induced genes, cold-shock proteins (Csps) are the most prominent. A csp-homologous gene, cspBPi, has been cloned from the Arctic bacterium Polaribacter irgensii KOPRI 22228, and overexpression of this gene greatly increased the freezing tolerance of its host. This protein consists of a unique N-terminal domain and a well conserved C-terminal cold shock domain. To elucidate the detailed mechanisms involved in the extraordinary freeze-tolerance conferred by CspBPi, we identified the responsible domain by mutational analysis. Changes of residues in the cold shock domain that are crucial for binding RNA or single-stranded DNA did not impair the ability of the host to survive freezing stress. All domain-shuffled CspBPi variants containing the N-terminal domain retained the ability to confer superior freeze-tolerance. Slow electrophoretic mobility and far-UV circular dichroism spectra of the N-terminal domain suggested an intrinsically disordered structure for this region. The N-terminal domain also bound to lipid vesicles in vitro. This lipid vesicle binding characteristic is shared with other intrinsically disordered proteins, such as α-synuclein and plant dehydrins, known to confer cold-tolerance when overexpressed, suggesting a mechanism for cold-survival through membrane binding.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance.

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

Zinc preconditioning protects against neuronal apoptosis through the mitogen-activated protein kinase-mediated induction of heat shock protein 70.

During brain ischemic preconditioning (PC), mild bursts of ischemia render neurons resistant to subsequent strong ischemic injuries. Previously, we reported that zinc plays a key role in PC-induced neuroprotection in vitro and in vivo. Zinc-triggered p75(NTR) induction transiently activates caspase-3, which cleaves poly(ADP-ribose) polymerase-1 (PARP-1). Subsequently, the PARP-1 over-activation-induced depletion of nicotinamide adenine dinucleotide (NAD(+))/adenosine triphosphate (ATP) after exposures to lethal doses of zinc or N-methyl-D-aspartate is significantly attenuated in cortical neuronal cultures. In the present study, zinc-mediated preconditioning (Zn PC) reduced apoptotic neuronal death that was caused by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), etoposide, or staurosporine in mouse cortical cells. We focused on heat shock protein 70 (HSP70) because NAD(+)/ATP depletion does not directly cause apoptosis, and HSP70 can inhibit the activation of caspase-9 or caspase-3 by preventing apoptosome formation or cytochrome C release. Zn PC-mediated HSP70 induction was required for neuroprotection against neuronal apoptosis, and geldanamycin-induced HSP70 induction sufficiently blocked neuronal apoptotic cell death. Furthermore, Zn PC-mediated HSP70 induction was blocked by chemical inhibitors of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase (MAPK) signaling, but not c-Jun N-terminal protein kinase. Similarly, neuroprotection by Zn PC against TPEN-induced apoptosis was almost completely reversed by the blockade of ERK or p38 MAPK signaling. Our findings suggest that the ERK- or p38 MAPK-mediated induction of HSP70 plays a key role in inhibiting caspase-3 activation during Zn PC.

Retarded protein folding of the human Z-type α1-antitrypsin variant is suppressed by Cpr2p.

The human Z-type α1-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α1-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α1-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α1-antitrypsin-expressing yeast cells compared to that in wild-type α1-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α1-antitrypsin increased significantly. The interaction between Z-type α1-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α1-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α1-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α1-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α1-antitrypsin.

Identification of chaperones in freeze tolerance in Saccharomyces cerevisiae.

Exposure to low temperatures reduces protein folding rates and induces the cold denaturation of proteins. Considering the roles played by chaperones in facilitating protein folding and preventing protein aggregation, chaperones must exist that confer tolerance to cold stress. Here, yeast strains lacking individual chaperones were screened for reduced freezing tolerance. In total, 19 of 82 chaperone-deleted strains tested were more sensitive to freeze-thaw treatment than wild-type cells. The reintroduction of the respective chaperone genes into the deletion mutants recovered the freeze tolerance. The freeze sensitivity of the chaperone-knockout strains was also retained in the presence of 20% glycerol.

α-Synuclein modulates neurite outgrowth by interacting with SPTBN1.

α-Synuclein is the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of surviving neuronal cells in Parkinson's disease patients. However, the physiological role played by α-synuclein remains unclear. In this study, spectrin beta non-erythrocyte 1 (SPTBN1) interacted with α-synuclein in phage display assays using a normalized human brain cDNA library. A direct interaction between α-synuclein and SPTBN1 was confirmed by GST pull-down and co-immunoprecipitation assays. SPTBN1 and α-synuclein proteins colocalized in N2a neuronal cells. Transfection of SPTBN1 caused human SH-SY5Y dopaminergic neuron cells to inappropriately induce neurites, which extended from cell bodies. Cotransfection with α-synuclein reversed SPTBN1-induced excessive neurite branching in SH-SY5Y cells, and only a single neurite extended from each neuron. These results suggest that α-synuclein modulates neurite outgrowth by interacting with cytoskeletal proteins such as SPTBN1.

Incidence rates and risk factors for vascular events in patients with essential thrombocythemia: a multicenter study from Korea.

BACKGROUND: Essential thrombocythemia (ET) is classified as a Philadelphia chromosome-negative classic myeloproliferative neoplasm. ET is a clonal stem cell disorder that is often associated with JAK2 mutations and shares phenotypic and pathogenetic similarities with other myeloproliferative neoplasms. Hemorrhagic complications and arterial and venous thrombosis are common in patients with ET. The aim of this retrospective analysis was to assess the cumulative incidence rate and risk factors for thrombohemorrhagic events in patients with ET based on a multicenter study in Korea. PATIENTS AND METHODS: A total of 239 patients with ET, from February 1995 to April 2011, were retrospectively analyzed from 4 Korean academic institutions. Data were collected through the review of medical records, and vascular events were confirmed by diagnostic procedures for establishing thrombosis and hemorrhagic complications. RESULTS: Of the patients (median age, 61 years; median follow-up, 51.8 months), 32 (13.4%) experienced thrombohemorrhagic complications. The 10-year cumulative incidence rate showed a 20.6% incidence of thrombohemorrhagic events. In univariate analysis, the presence of JAK2 mutations, high-risk group, previous thrombohemorrhagic events, and >60 years old were shown to have higher incidences of vascular events than any other factors. In multivariate analysis, previous thrombotic events and JAK2 mutations were independent risk factors for vascular events (hazard ratio, 2.907 [95% CI, 1.142-7.406], P =.025; and 4.146 [95% CI 1.227-14.018], P = 0.022). CONCLUSION: Previous thrombotic history and the JAK2 V617F mutation were associated with a higher 10-year cumulative incidence rate of thrombohemorrhagic events.

Human α-synuclein modulates vesicle trafficking through its interaction with prenylated Rab acceptor protein 1.

α-Synuclein has been implicated in the pathogenesis of Parkinson's disease. Although it is highly conserved, its physiological function has not yet been elucidated in detail. In an effort to define the function of α-synuclein, interacting proteins were screened in phage display assays. Prenylated Rab acceptor protein 1 (PRA1) was identified as an interacting partner. A selective interaction between α-synuclein and PRA1 was confirmed by coimmunoprecipitation and GST pull-down assays. PRA1 and α-synuclein were colocalized in N2a neuronal cells. Cotransfection of α-synuclein and PRA1 caused vesicles to accumulate in the periphery of the cytosol in neuronal cells, suggesting that overexpression of α-synuclein hinders proper vesicle trafficking and recycling as a result of the interaction between α-synuclein and PRA1.

Zinc-triggered induction of tissue plasminogen activator by brain-derived neurotrophic factor and metalloproteinases.

Tissue plasminogen activator (tPA) is necessary for hippocampal long-term potentiation. Synaptically released zinc also contributes to long-term potentiation, especially in the hippocampal CA3 region. Using cortical cultures, we examined whether zinc increased the concentration and/or activity of tPA. Two hours after a 10-min exposure to 300 µM zinc, expression of tPA and its substrate, plasminogen, were significantly increased, as was the proteolytic activity of tPA. In contrast, increasing extracellular or intracellular calcium levels did not affect the expression or secretion of tPA. Changing zinc influx or chelating intracellular zinc also failed to alter tPA/plasminogen induction by zinc, indicating that zinc acts extracellularly. Zinc-mediated extracellular activation of matrix metalloproteinase (MMP) underlies the up-regulation of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase (Trk) signaling. Consistent with these findings, co-treatment with a neutralizing antibody against BDNF or specific inhibitors of MMPs or Trk largely reversed tPA/plasminogen induction by zinc. Treatment of cortical cultures with p-aminophenylmercuric acetate, an MMP activator, MMP-2, or BDNF alone induced tPA/plasminogen expression. BDNF mRNA and protein expression was also increased by zinc and mediated by MMPs. Thus, an extracellular zinc-dependent, MMP- and BDNF-mediated synaptic mechanism may regulate the levels and activity of tPA.

The hexapeptide PGVTAV suppresses neurotoxicity of human α-synuclein aggregates.

In Parkinson's disease patients, α-synuclein is the major component of the intracellular protein aggregates found in dopaminergic neurons. Previously, short synthetic α-synuclein-derived peptides have been shown to not only prevent α-synuclein fibrillation but also dissolve preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV was the shortest peptide that retained the ability to block α-synuclein fibrillation. For preventative or therapeutic effectiveness, a treatment must suppress the neurotoxicity of α-synuclein aggregates and remain stable in plasma. The present study shows that specific peptides can protect neuronal cells from α-synuclein aggregation-induced cell death. The ß-sheet-breaking hexapeptide PGVTAV remained intact in human plasma for longer than one day, suggesting that it may be a candidate for the development of therapeutics to treat Parkinson's disease.

Herpesviridae viral infections following rituximab combined chemotherapy in patients with diffuse large B-cell lymphoma.

BACKGROUND: Herpesviridae viral infections (HVIs) are particularly common in patients with hematologic malignancies after undergoing hematopoietic stem cell transplantation or receiving chemotherapy. However, there have been few reports on the incidence and risk factors of HVIs in diffuse large B-cell lymphoma (DLBL) patients treated with rituximab combined chemotherapy. METHODS: We analyzed 270 patients who were newly diagnosed with DLBL. All of the patients had received rituximab combined chemotherapy between June 2004 and April 2010. RESULTS: Twenty-nine patients (10.7%) developed HVI a median of 5.57 months (range 0.37-30.03) after initial chemotherapy. The estimated cumulative incidence rates of HVIs were 8.3 and 12.8% at 1 and 3 years, respectively, in all patients. Independent risk factors for HVIs were a high international prognostic index risk [p = 0.017, hazard ratio (HR) 2.633, 95% confidence interval (CI) 1.185-5.850], neutropenic fever (p = 0.023, HR 2.476, 95% CI 1.134-5.406) and a high cumulative dose of steroids (p = 0.023, HR 2.921, 95% CI 1.162-7.346). CONCLUSION: A high international prognostic index risk, neutropenic fever and a high cumulative dose of steroids appear to be risk factors for HVI in DLBL patients who are undergoing rituximab combined chemotherapy.

Overexpression of cold shock protein A of Psychromonas arctica KOPRI 22215 confers cold-resistance.

A polar bacterium was isolated from Arctic sea sediments and identified as Psychromonas artica, based on 16S rDNA sequence. Psychromonas artica KOPRI 22215 has an optimal growth temperature of 10 degrees C and a maximum growth temperature of 25 degrees C, suggesting this bacterium is a psychrophile. Cold shock proteins (Csps) are induced upon temperature downshift by more than 10 degrees C. Functional studies have researched mostly Csps of a mesophilic bacterium Escherichia coli, but not on those of psychrophilic bacteria. In an effort to understand the molecular mechanisms of psychrophilic bacteria that allow it withstand freezing environments, we cloned a gene encoding a cold shock protein from P. artica KOPRI 22215 (CspA(Pa)) using the conserved sequences in csp genes. The 204 bp-long ORF encoded a protein of 68 amino acids, sharing 56% homology to previously reported E. coli CspA protein. When CspA(Pa) was overexpressed in E. coli, it caused cell growth-retardation and morphological elongation. Interestingly, overexpression of CspA(Pa) drastically increased the host's cold-resistance by more than ten times, suggesting the protein aids survival in polar environments.

Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228.

Exposure to low temperatures induces the biosynthesis of specific sets of proteins, including cold shock proteins (Csps). Since many of the specific functions of pychrophilic Csps are unknown, the roles of Csps from an Arctic bacterium, Polaribacter irgensii KOPRI 22228, were examined. The genes encoding CspA and CspC of P. irgensii were cloned in this study. Sequence analysis showed that these proteins have cold shock domains containing two RNA-binding motifs, RNP1 and RNP2. Both proteins bound oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. When the P. irgensii Csps were overexpressed in Escherichia coli, the cold-resistance of the host was increased by more than five-fold. The P. irgensii Csps also rescued a cold-sensitive E. coli csp-quadruple deletion strain, BX04, at low temperatures. These results suggest that Csps from P. irgensii play a role in survival in polar environments.

beta-Sheet-breaking peptides inhibit the fibrillation of human alpha-synuclein.

alpha-Synuclein is the major components of the intracellular protein-aggregates, found in the dopaminergic neurons of Parkinson's disease patients. Previously, we screened for alpha-synuclein substitution mutants that prevent fibril formation of both wild-type and Parkinson's disease-linked alpha-synuclein variants. In the present study, we show that short synthetic peptides derived from these mutant sequences not only prevented alpha-synuclein fibrillation but also dissolved preformed alpha-synuclein aggregates in vitro. The hexapeptide PGVTAV, which was the shortest peptide that retained the ability to block alpha-synuclein fibrillation, may serve as a lead compound for the development of therapeutics for Parkinson's disease.

Aggregation-defective alpha-synuclein mutants inhibit the fibrillation of Parkinson's disease-linked alpha-synuclein variants.

Alpha-synuclein comprises the fibrillar core of Lewy bodies, which is one of the histologically defining lesions of Parkinson's disease. Previously, we screened for alpha-synuclein substitution mutants that do not form fibrils. For preventative or therapeutic uses, it is essential to suppress the oligomerization/fibrillation of the wild-type and PD-linked alpha-synuclein proteins. Here we have examined the effects of fibrillation-retarded alpha-synuclein mutants on fibril formation by wild-type and PD-linked alpha-synuclein molecules. Six self-aggregation-defective alpha-synuclein mutants completely inhibit the fibrillation of both wild-type and Parkinson's disease-linked alpha-synuclein variants. These results suggest future applications for gene therapy: the transplantation of a fibrillation-blocking mutant alpha-synuclein gene into individuals who carry an early-onset PD-associated alpha-synuclein allele. Short synthetic peptides derived from these mutant sequences may also serve as a lead compound for the development of therapeutics for Parkinson's disease.

Identification and expression of the tig gene coding for trigger factor from psychrophilic bacteria with no information of genome sequence available.

Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis-trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15 degrees C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4 degrees C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.