Publications by authors named "James G Omichinski"

56 Publications

Role of active site arginine residues in substrate recognition by PPM1A.

Biochem Biophys Res Commun 2021 Oct 6;581:1-5. Epub 2021 Oct 6.

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, 060-0810, Japan. Electronic address:

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.
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http://dx.doi.org/10.1016/j.bbrc.2021.10.001DOI Listing
October 2021

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mol Cell 2021 09;81(18):3848-3865.e19

CRCHUM, 900 Saint-Denis St, Montréal, QC H2X 0A9, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada. Electronic address:

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
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http://dx.doi.org/10.1016/j.molcel.2021.08.028DOI Listing
September 2021

Pax7 pioneer factor action requires both paired and homeo DNA binding domains.

Nucleic Acids Res 2021 07;49(13):7424-7436

Laboratory of Molecular Genetics, Institut de recherches cliniques de Montréal (IRCM), Montréal QC H2W 1R7, Canada.

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.
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http://dx.doi.org/10.1093/nar/gkab561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8287922PMC
July 2021

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells.

Exp Hematol 2020 08 16;88:68-82.e5. Epub 2020 Jul 16.

Maisonneuve-Rosemont Hospital Research Center, University of Montreal, CIUSSS de l'Est-de-l'Île de Montréal, Montreal, QC, Canada; Department of Medicine, University of Montréal, Montréal, QC, Canada. Electronic address:

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.
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http://dx.doi.org/10.1016/j.exphem.2020.07.004DOI Listing
August 2020

Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors.

Pharmacol Ther 2020 11 7;215:107622. Epub 2020 Jul 7.

Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. Electronic address:

Protein phosphatases and kinases control multiple cellular events including proliferation, differentiation, and stress responses through regulating reversible protein phosphorylation, the most important post-translational modification. Members of metal-dependent protein phosphatase (PPM) family, also known as PP2C phosphatases, are Ser/Thr phosphatases that bind manganese/magnesium ions (Mn/Mg) in their active center and function as single subunit enzymes. In mammals, there are 20 isoforms of PPM phosphatases: PPM1A, PPM1B, PPM1D, PPM1E, PPM1F, PPM1G, PPM1H, PPM1J, PPM1K, PPM1L, PPM1M, PPM1N, ILKAP, PDP1, PDP2, PHLPP1, PHLPP2, PP2D1, PPTC7, and TAB1, whereas there are only 8 in yeast. Phylogenetic analysis of the DNA sequences of vertebrate PPM isoforms revealed that they can be divided into 12 different classes: PPM1A/PPM1B/PPM1N, PPM1D, PPM1E/PPM1F, PPM1G, PPM1H/PPM1J/PPM1M, PPM1K, PPM1L, ILKAP, PDP1/PDP2, PP2D1/PHLPP1/PHLPP2, TAB1, and PPTC7. PPM-family members have a conserved catalytic core region, which contains the metal-chelating residues. The different isoforms also have isoform specific regions within their catalytic core domain and terminal domains, and these regions may be involved in substrate recognition and/or functional regulation of the phosphatases. The twenty mammalian PPM phosphatases are involved in regulating diverse cellular functions, such as cell cycle control, cell differentiation, immune responses, and cell metabolism. Mutation, overexpression, or deletion of the PPM phosphatase gene results in abnormal cellular responses, which lead to various human diseases. This review focuses on the structures and biological functions of the PPM-phosphatase family and their associated diseases. The development of specific inhibitors against the PPM phosphatase family as a therapeutic strategy will also be discussed.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107622DOI Listing
November 2020

Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.

Structure 2020 05 28;28(5):573-585.e5. Epub 2020 Apr 28.

Département de Biochimie et Médicine Moléculaire, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada. Electronic address:

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.
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http://dx.doi.org/10.1016/j.str.2020.04.002DOI Listing
May 2020

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner.

Structure 2020 02 23;28(2):157-168.e5. Epub 2019 Dec 23.

Département de Biochimie et Médicine Moléculaire, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada. Electronic address:

The interactions between SUMO proteins and SUMO-interacting motif (SIM) in nuclear bodies formed by the promyelocytic leukemia (PML) protein (PML-NBs) have been shown to be modulated by either phosphorylation of the SIMs or acetylation of SUMO proteins. However, little is known about how this occurs at the atomic level. In this work, we examined the role that acetylation of SUMO1 plays on its binding to the phosphorylated SIMs (phosphoSIMs) of PML and Daxx. Our results demonstrate that SUMO1 binding to the phosphoSIM of either PML or Daxx is dramatically reduced by acetylation at either K39 or K46. However, acetylation at K37 only impacts binding to Daxx. Structures of acetylated SUMO1 variants bound to the phosphoSIMs of PML and Daxx demonstrate that there is structural plasticity in SUMO-SIM interactions. The plasticity observed in these structures provides a robust mechanism for regulating SUMO-SIM interactions in PML-NBs using signaling generated post-translational modifications.
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http://dx.doi.org/10.1016/j.str.2019.11.019DOI Listing
February 2020

The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein.

Biochem Biophys Res Commun 2020 01 2;521(3):681-686. Epub 2019 Nov 2.

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, 060-0810, Japan. Electronic address:

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.130DOI Listing
January 2020

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension.

BMC Biotechnol 2018 12 6;18(1):76. Epub 2018 Dec 6.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, H3C 3J7, QC, Canada.

Background: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k (7.2 ± 0.5 min) and K (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k and K values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.
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http://dx.doi.org/10.1186/s12896-018-0485-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282390PMC
December 2018

The type IV secretion system core component VirB8 interacts via the β1-strand with VirB10.

FEBS Lett 2017 08 11;591(16):2491-2500. Epub 2017 Aug 11.

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, QC, Canada.

In this work, we provide evidence for the interactions between VirB8 and VirB10, two core components of the type IV secretion system (T4SS). Using nuclear magnetic resonance experiments, we identified residues on the β1-strand of Brucella VirB8 that undergo chemical shift changes in the presence of VirB10. Bacterial two-hybrid experiments confirm the importance of the β1-strand, whereas phage display experiments suggest that the α2-helix of VirB8 may also contribute to the interaction with VirB10. Conjugation assays using the VirB8 homolog TraE as a model show that several residues on the β1-strand of TraE are important for T4SS function. Together, our results suggest that the β1-strand of VirB8-like proteins is essential for their interaction with VirB10 in the T4SS complex.
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http://dx.doi.org/10.1002/1873-3468.12770DOI Listing
August 2017

Oligomerization enhances the binding affinity of a silver biomineralization peptide and catalyzes nanostructure formation.

Sci Rep 2017 05 3;7(1):1400. Epub 2017 May 3.

Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, 060-0810, Japan.

Binding affinity and specificity are crucial factors that influence nanostructure control by biomineralization peptides. In this paper, we analysed the role that the oligomeric state of a silver biomineralization peptide plays in regulating the morphology of silver nanostructure formation. Oligomerization was achieved by conjugating the silver specific TBP biomineralization peptide to the p53 tetramerization domain peptide (p53Tet). Interestingly, the TBP-p53Tet tetrameric peptide acted as a growth catalyst, controlling silver crystal growth, which resulted in the formation of hexagonal silver nanoplates without consuming the peptide. The TBP-p53Tet peptide caps the surface of the silver crystals, which enhances crystal growth on specific faces and thereby regulates silver nanostructure formation in a catalytic fashion. The present findings not only provide an efficient strategy for controlling silver nanostructure formation by biomineralization peptides, but they also demonstrate that in this case the oligomeric peptides play a unique catalytic role.
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http://dx.doi.org/10.1038/s41598-017-01442-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431226PMC
May 2017

Structural characterization of interactions between transactivation domain 1 of the p65 subunit of NF-κB and transcription regulatory factors.

Nucleic Acids Res 2017 May;45(9):5564-5576

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3C 3J7, Canada.

p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65-p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAFII31. Like other acidic TADs, the p65 TAD contains two subdomains (p65TA1 and p65TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the ΦXXΦΦ motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.
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http://dx.doi.org/10.1093/nar/gkx146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435986PMC
May 2017

Monomer-to-dimer transition of Brucella suis type IV secretion system component VirB8 induces conformational changes.

FEBS J 2017 04 23;284(8):1218-1232. Epub 2017 Mar 23.

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.

Secretion systems are protein complexes essential for bacterial virulence and potential targets for antivirulence drugs. In the intracellular pathogen Brucella suis, a type IV secretion system mediates the translocation of virulence factors into host cells and it is essential for pathogenicity. VirB8 is a core component of the secretion system and dimerization is important for functionality of the protein complex. We set out to study dimerization and possible conformational changes of VirB8 from B. suis (VirB8s) using nuclear magnetic resonance, X-ray crystallography, and differential scanning fluorimetry. We identified changes of the protein induced by a concentration-dependent monomer-to-dimer transition of the periplasmic domain (VirB8sp). We also show that the presence of the detergent CHAPS alters several signals in the heteronuclear single quantum coherence (HSQC) spectra and some of these chemical shift changes correspond to those observed during monomer-dimer transition. X-ray analysis of a monomeric variant (VirB8sp ) demonstrates that significant structural changes occur in the protein's α-helical regions (α2 and α4). We localized chemical shift changes of residues at the dimer interface as well as to the α1 helix that links this interface to a surface groove that binds dimerization inhibitors. Fragment-based screening identified small molecules that bind to VirB8sp and two of them have differential binding affinity for wild-type and the VirB8sp variant underlining their different conformations. The observed chemical shift changes suggest conformational changes of VirB8s during monomer-dimer transition that may play a role during secretion system assembly or function and they provide insights into the mechanism of inhibitor action.

Database: BMRB accession no. 26852 and PDB 5JBS.
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http://dx.doi.org/10.1111/febs.14049DOI Listing
April 2017

Molecular basis of interactions between SH3 domain-containing proteins and the proline-rich region of the ubiquitin ligase Itch.

J Biol Chem 2017 04 24;292(15):6325-6338. Epub 2017 Feb 24.

From the Departments of Biological Sciences and

The ligase Itch plays major roles in signaling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding are critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pulldown assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with endophilins, amphyphisins, and pacsins but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modeling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR toward its most common SH3 domain-containing partners and demonstrate that the PRR region is sufficient for binding.
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http://dx.doi.org/10.1074/jbc.M116.754440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391761PMC
April 2017

Structural and Biochemical Characterization of Organotin and Organolead Compounds Binding to the Organomercurial Lyase MerB Provide New Insights into Its Mechanism of Carbon-Metal Bond Cleavage.

J Am Chem Soc 2017 01 3;139(2):910-921. Epub 2017 Jan 3.

Département de Biochimie et Médicine Moléculaire, Université de Montréal , Montréal, Quebec H3C 3J7 Canada.

The organomercurial lyase MerB has the unique ability to cleave carbon-Hg bonds, and structural studies indicate that three residues in the active site (C96, D99, and C159 in E. coli MerB) play important roles in the carbon-Hg bond cleavage. However, the role of each residue in carbon-metal bond cleavage has not been well-defined. To do so, we have structurally and biophysically characterized the interaction of MerB with a series of organotin and organolead compounds. Studies with two known inhibitors of MerB, dimethyltin (DMT) and triethyltin (TET), reveal that they inhibit by different mechanisms. In both cases the initial binding is to D99, but DMT subsequently binds to C96, which induces a conformation change in the active site. In contrast, diethyltin (DET) is a substrate for MerB and the Sn product remains bound in the active site in a coordination similar to that of Hg following cleavage of organomercurial compounds. The results with analogous organolead compounds are similar in that trimethyllead (TML) is not cleaved and binds only to D99, whereas diethyllead (DEL) is a substrate and the Pb product remains bound in the active site. Binding and cleavage is an exothermic reaction, while binding to D99 has negligible net heat flow. These results show that initial binding of organometallic compounds to MerB occurs at D99 followed, in some cases, by cleavage and loss of the organic moieties and binding of the metal ion product to C96, D99, and C159. The N-terminus of MerA is able to extract the bound Pb but not the bound Sn. These results suggest that MerB could be utilized for bioremediation applications, but certain organolead and organotin compounds may present an obstacle by inhibiting the enzyme.
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http://dx.doi.org/10.1021/jacs.6b11327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040655PMC
January 2017

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

Biochemistry 2016 Feb 11;55(7):1070-81. Epub 2016 Feb 11.

Department of Chemistry, Dartmouth College , Hanover, New Hampshire 03755, United States.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.
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http://dx.doi.org/10.1021/acs.biochem.5b01298DOI Listing
February 2016

Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

Biochemistry 2015 Dec 10;54(50):7345-54. Epub 2015 Dec 10.

Department of Biological Sciences and ‡Department of Biochemistry and Molecular Medicine, University of Montreal , Montreal, Quebec H3C 3J7, Canada.

Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested.
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http://dx.doi.org/10.1021/acs.biochem.5b01131DOI Listing
December 2015

A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

Proc Natl Acad Sci U S A 2015 May 27;112(19):6021-6. Epub 2015 Apr 27.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada H3C 3J7; and

Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.
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http://dx.doi.org/10.1073/pnas.1503688112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434773PMC
May 2015

Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

Structure 2015 Jan 11;23(1):126-138. Epub 2014 Dec 11.

Département de Biochimie et Médicine Moléculaire, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada. Electronic address:

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.
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http://dx.doi.org/10.1016/j.str.2014.10.015DOI Listing
January 2015

Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

Methods Enzymol 2014 ;549:49-84

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada. Electronic address:

Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest.
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http://dx.doi.org/10.1016/B978-0-12-801122-5.00003-9DOI Listing
July 2015

Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.

PLoS Pathog 2014 Mar 27;10(3):e1004042. Epub 2014 Mar 27.

Département de Biochimie et Médicine Moléculaire, Université de Montréal, Succursale Centre-Ville, Montréal, Québec, Canada.

Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.
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http://dx.doi.org/10.1371/journal.ppat.1004042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968163PMC
March 2014

Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation.

J Biol Chem 2013 Dec 30;288(51):36312-27. Epub 2013 Oct 30.

From the Département de Biochimie, Université de Montréal, C. P. 6128 Succursale Centre-Ville, Montréal, Quebec H3C 3J7, Canada.

Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.
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http://dx.doi.org/10.1074/jbc.M113.486845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868746PMC
December 2013

Structural characterization of a noncovalent complex between ubiquitin and the transactivation domain of the erythroid-specific factor EKLF.

Structure 2013 Nov 17;21(11):2014-24. Epub 2013 Oct 17.

Département de Biochimie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.

Like other acidic transactivation domains (TAD), the minimal TAD from the erythroid-specific transcription factor EKLF (EKLFTAD) has been shown to contribute both to its transcriptional activity as well as to its ubiquitin(UBI)-mediated degradation. In this article, we examine the activation-degradation role of the acidic TAD of EKLF and demonstrate that the first 40 residues (EKLFTAD1) within this region form a noncovalent interaction with UBI. Nuclear magnetic resonance (NMR) structural studies of an EKLFTAD1-UBI complex show that EKLFTAD1 adopts a 14-residue α helix that forms the recognition interface with UBI in a similar manner as the UBI-interacting helix of Rabex5. We also identify a similar interaction between UBI and the activation-degradation region of SREBP1a, but not with the activation-degradation regions of p53, GAL4, and VP16. These results suggest that select activation-degradation regions like the ones found in EKLF and SREBP1a function in part through their ability to form noncovalent interactions with UBI.
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http://dx.doi.org/10.1016/j.str.2013.08.027DOI Listing
November 2013

An experimentally tested scenario for the structural evolution of eukaryotic Cys2His2 zinc fingers from eubacterial ros homologs.

Mol Biol Evol 2013 Jul 9;30(7):1504-13. Epub 2013 Apr 9.

Dipartimento di Scienze Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli , Via Vivaldi 43, 81100 Caserta, Italy.

The exact evolutionary origin of the zinc finger (ZF) domain is unknown, as it is still not clear from which organisms it was first derived. However, the unique features of the ZF domains have made it very easy for evolution to tinker with them in a number of different manners, including their combination, variation of their number by unequal crossing-over or tandem duplication and tuning of their affinity for specific DNA sequence motifs through point substitutions. Classical Cys2His2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys2His2 ZF domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens and recently characterized. The present work focuses on the evolution of the classical ZF domains with the goal of trying to determine whether eukaryotic ZFs have evolved from the prokaryotic Ros-like proteins. Our results, based on computational and experimental data, indicate that a single insertion of three amino acids in the short loop that separates the β-sheet from the α-helix of the Ros protein is sufficient to induce a structural transition from a Ros like to an eukaryotic-ZF like structure. This observation provides evidence for a structurally plausible and parsimonious scenario of fold evolution, giving a structural basis to the hypothesis of a horizontal gene transfer (HGT) from bacteria to eukaryotes.
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http://dx.doi.org/10.1093/molbev/mst068DOI Listing
July 2013

Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER.

Nucleic Acids Res 2013 Feb 7;41(4):2736-45. Epub 2013 Jan 7.

Département de Biochimie, Université de Montréal C.P. 6128 Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.

XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4-Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4-Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription.
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http://dx.doi.org/10.1093/nar/gks1321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575800PMC
February 2013

Preparation of λN-GST fusion protein for affinity immobilization of RNA.

Methods Mol Biol 2012 ;941:123-35

Département de Biochimie, Université de Montréal, Montreal, QC, Canada.

Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses.
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http://dx.doi.org/10.1007/978-1-62703-113-4_10DOI Listing
March 2013

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

Nucleic Acids Res 2012 Jul 28;40(12):5739-50. Epub 2012 Feb 28.

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.
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http://dx.doi.org/10.1093/nar/gks194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384317PMC
July 2012

Inhibition of human papillomavirus DNA replication by an E1-derived p80/UAF1-binding peptide.

J Virol 2012 Apr 25;86(7):3486-500. Epub 2012 Jan 25.

Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada.

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target.
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http://dx.doi.org/10.1128/JVI.07003-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302507PMC
April 2012

Structure-based design of a potent artificial transactivation domain based on p53.

J Am Chem Soc 2012 Jan 12;134(3):1715-23. Epub 2012 Jan 12.

Département de Biochimie, Université de Montréal, C.P. 6128 Succursale, Centre-Ville, Montréal, Quebec H3C 3J7, Canada.

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.
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http://dx.doi.org/10.1021/ja208999eDOI Listing
January 2012
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