Multiple Structural and Epigenetic Defects in the Human Leukocyte Antigen class I Antigen Presentation Pathway in a Recurrent Metastatic Melanoma Following Immunotherapy [Immunology]

September 17th, 2015 by

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study, we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis following immunotherapy. These defects include loss of tapasin and one HLA haplotype, as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germline frame-shift mutation in exon 3 (TAPBP684delA) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible following DNA methyltransferase 1 depletion. This treatment, combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.
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Structural basis for antigenic peptide recognition and processing by ER aminopeptidase 2 [Protein Structure and Folding]

September 17th, 2015 by

Endoplasmic reticulum aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5Å and 2.7Å respectively and compared them to the apo form structure determined to 3.0Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center towards the distal end of the internal cavity making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product although decreasing electron density towards its C-terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C-terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

Integrin {alpha}6{beta}4 Promotes Autocrine EGFR Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF) [Gene Regulation]

September 17th, 2015 by

Integrin α6β4 is upregulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 upregulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in the TCGA pancreatic cancer database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on HGF-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways including MAPK, PI3K and PKC. Moreover, MMP activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional (3D) culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional upregulation of key EGFR family members, and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.
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Oligomerization and Membrane-Binding Properties of Covalent Adducts Formed by the Interaction of Alpha-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL) [Molecular Bases of Disease]

September 17th, 2015 by

Oxidative deamination of dopamine (DA) produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post mortem brains of Parkinson′s disease (PD) patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for DA metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers which may be crosslinked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipild-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.
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ATDC (Ataxia Telangectasia Group D Complementing) Promotes Radioresistance Through an Interaction with the RNF8 Ubiquitin Ligase [DNA and Chromosomes]

September 17th, 2015 by

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The Ataxia-Telangiectasia group D Complementing gene (ATDC, also called TRIM29), is highly expressed in many malignancies. It participates in the DNA damage response (DDR) downstream of ATM and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (aa 348-588) of ATDC and the ring domain of RNF8 and was disrupted by mutation of ATDC S550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.
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Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-Molecule Chromatin Immunoprecipitation Imaging [DNA and Chromosomes]

September 17th, 2015 by

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical to appreciate the initiation, propagation and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied, but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem (mES) cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Further, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric while PRC2 can dimerize in the nucleoplasm. We showed that ES cell-differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin, but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.
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WNT10B Enhances Proliferation Through {beta}-catenin and RAC1 GTPase in Human Corneal Endothelial Cells [Signal Transduction]

September 14th, 2015 by Lee, J. G., Heur, M.

The cornea is the anterior, transparent tissue of the human eye that serves as its main refractive element. The corneal endothelial cells are arranged as a monolayer on the posterior surface of the cornea and function as a pump to counteract the leakiness of its basement membrane. Maintaining the cornea in a slightly dehydrated state is critical for maintenance of corneal transparency. Adult human corneal endothelial cells are G1 arrested, even in response to injury, leading to an age-dependent decline in the endothelial cell density. Corneal edema and subsequent vision loss ensues when the endothelial cell density decreases below a critical threshold. Vision loss secondary to corneal endothelial dysfunction is a common indication for transplantation in developed nations. An impending increase in demand for and a current global shortage of donor corneas will necessitate development of treatments for vision loss due to endothelial dysfunction that does not rely on donor corneas. Wnt ligands regulate many critical cellular functions such as proliferation, making them attractive candidates for modulation in corneal endothelial dysfunction. We show WNT10B causes nuclear transport and binding of RAC1 and β-catenin in human corneal endothelial cells, leading to activation of Cyclin D1 expression and proliferation. Our findings indicate WNT10B promotes proliferation in human corneal endothelial cells by simultaneously utilizing both β-catenin dependent and independent pathways, and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.

Posttranslational Regulation of Human DNA Polymerase {iota} [Enzymology]

September 14th, 2015 by

Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via K11- and K48- linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se.

Pore Hydration States of KcsA Potassium Channels in Membranes [Lipids]

September 14th, 2015 by

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the trans-membrane domain of M2 protein of Influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid-state NMR. In contrast, the M2 proton channel shows a "V"- shaped water profile across the membrane, with a narrow constriction at the center, like the hour-glass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.

Two Hydrophobic Residues Can Determine The Specificity Of MAP Kinase Docking Interactions [Cell Biology]

September 14th, 2015 by Bardwell, A. J., Bardwell, L.

Mitogen-activated protein kinases (MAPKs) bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK and p38) can bind selectively to D-sites in their authentic substrates and regulators, while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just one or two key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or visa versa. These specificity-determining differences are also found in the D-sites of the ETS-family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK vs. JNK as predicted. These results provide new insights into docking specificity, and suggest that this specificity can evolve rapidly by changes to just one or two amino acids.