Laccases are multicopper enzymes that catalyze oxidation of electron-rich substrates coupled to reduction of molecular oxygen to water. Since the Type 1 copper (T1 Cu) is the site where electrons are ...withdrawn from the substrate, it is assumed that the reduction potential of this copper correlates with enzyme activity. Herein, we studied the correlation of the T1 Cu reduction potential and the enzymatic activity of the small two-domain laccase Ssl1 from Streptomyces sviceus. For a systematic approach, we aimed to minimize any effects other than the reduction potential difference. To this end, we constructed a series of Ssl1 mutants with reduction potentials varying from <290 to 560 mV. Along with the hydrophobicity of the axial ligand of the T1 Cu also structural changes in the substrate binding site and additional hydrogen bonding increased the reduction potential. Enzyme activity experiments demonstrated that the T1 Cu reduction potential has a different effect on oxidation of different substrates. Whereas there was no obvious correlation between the T1 Cu reduction potential and kinetic parameters for the oxidation of syringaldazine (with a reduction potential of 390 mV), a good correlation was observed between the T1 Cu reduction potential and the conversion of substituted phenols with reduction potentials between 660 and 820 mV. This correlation was pronounced for the Ssl1 variants with reduction potentials above 470 mV, which demonstrated increased activities also during the oxidation of two dyes, alizarin red S and indigo carmine.
How the environment of the Type 1 copper of a laccase influences the enzyme activity remains an intriguing question. In this work, we constructed mutants of a small laccase from Streptomyces sviceus with reduction potentials ranging from <290 to 560 mV, and evaluated their activity towards high- and low-potential substrates. Display omitted
•Mutants of Ssl1 laccase with reduction potentials between <290 and 560 mV were constructed.•Reduction potential of the Type 1 copper (T1 Cu) correlated with the axial ligand hydrophobicity.•Activity towards substituted phenols correlated with the T1 Cu reduction potential.•Ssl1 variant with a reduction potential of 560 mV oxidized substrates not accepted by wild type.
Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show ...here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.
Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this ...often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.
BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP ...and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The over-expression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.
Mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition
. However, these inhibitors alone do not lead to tumor ...regression
, indicating the need for combination therapy.
high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members.
validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway.
combination of trametinib with BCL-2 inhibitors led to tumor inhibition in
-mutant and
-deleted xenografts.
Together, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients.
To examine the outcomes of patients with histologically confirmed central neurocytomas.
The data from 45 patients with central neurocytomas diagnosed between 1971 and 2003 were retrospectively ...evaluated. Various combinations of surgery, radiotherapy (RT), and chemotherapy had been used for treatment.
The median follow-up was 10.0 years. The 10-year overall survival and local control rate was 83% and 60%, respectively. Patients whose tumor had a mitotic index of <3 (per 10 high-power fields) experienced a 10-year survival and local control rate of 89% and 74%, respectively, compared with 57% (p = 0.040) and 46% (p = 0.14) for patients with a tumor mitotic index of > or =3. The 10-year survival and local control rate was 90% and 74% for patients with typical tumors compared with 63% (p = 0.055) and 46% (p = 0.41) for those with atypical tumors. A comparison of gross total resection with subtotal resection showed no significant difference in survival or local control. Postoperative RT improved local control at 10 years (75% with RT vs. 51% without RT, p = 0.045); however, this did not translate into a survival benefit. No 1p19q deletions were found in the 19 tumors tested.
Although the overall prognosis is quite favorable, one-third of patients experienced tumor recurrence or progression at 10 years, regardless of the extent of the initial resection. Postoperative RT significantly improved local control but not survival, most likely because of the effectiveness of salvage RT. For incompletely resected atypical tumors and/or those with a high mitotic index, consideration should be given to adjuvant RT because of the more aggressive nature.
Abstract The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, ...which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing that targeted knock down with shRNA’s triggers an apoptotic response through mitotic catastrophe. We now tested YM155, a novel small molecule selective BIRC5 suppressant that is currently in phase I/II clinical trials. Drug response curves showed IC50 values in the low nM range (median: 35 nM, range: 0.5–>10,000 nM) in a panel of 23 neuroblastoma cell lines and four TIC-lines, which resulted from an apoptotic response. Nine out of 23 cell lines were relatively resistant to YM155 with IC50 values >200 nM, although in the same cells shRNA mediated knock down of BIRC5 caused massive apoptosis. Analysis of differentially expressed genes between five most sensitive and five most resistant cell lines using Affymetrix mRNA expression data revealed ABCB1 (MDR1) as the most predictive gene for resistance to YM155. Inhibition of the multi-drug resistance pump ABCB1 with cyclosporine or knockdown with shRNA prior to treatment with YM155 demonstrated that cell lines with ABCB1 expression became 27–695 times more sensitive to YM155 treatment. We conclude that most neuroblastoma cell lines are sensitive to YM155 in the low nM range and that resistant cells can be sensitised by ABCB1 inhibitors. Therefore YM155 is a promising novel compound for treatment of neuroblastoma with low ABCB1 expression.
The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with ...delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.
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•HLA class I variants drive selection pressures on Gag to limit epitope production•The strength of the selective pressure is positively correlated to HLA frequencies•HIV adaptation to limit epitope production occurs at subtype-specific HIV motifs•HIV adapts in a predictable way to HLA frequencies in newly infected populations
CD8+ T cell responses against HIV-1 effectively delay disease progression in a minority of patients with relatively rare HLA variants but are ineffective in most. Here, Tenzer et al. identify fundamental HIV-1 adaptation to the conserved human antigen-processing machinery that feeds epitopes to HLA. This adaptation occurs at subtype-specific motifs, facilitates subtype diversification, is predictable, and results in CD8 epitope abundances that correlate inversely with the HLA allele frequencies in affected populations. Thus, HIV vaccine immunogenicity might be increased by unnatural substitutions at subtype-specific motifs.
Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with ...enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4–14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.
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•Proteasomal activity creates many length variants of the protective KK10 HIV-epitope•All KK10 length variants bind HLA-B∗27:05 through a conserved N-terminal motif•Short KK10 length variants outcompete immunogenic, longer variants for HLA binding•Epitope variant competition balances protective immune responses and viral escape
Pymm et al. investigate how HIV-1 adapts to intracellular epitope processing preferences to escape cellular immune responses. Using the processing of the HLA-B∗27:05-restricted epitope KK10 as a model, they find that processing generates several epitope length variants and demonstrate that peptide competition can dampen cellular immune responses in the context of HLA-B∗27:05.
Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been ...identified in neuroblastoma.
Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform ( http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation.
We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines.
Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.
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