A Simple Nickel Metal-Organic Framework-Catalyzed Borylation of Aryl Chlorides and Bromides.

We report a recyclable and efficient catalyst system based on a nickel-benzene tricarboxylic acid metal-organic framework (Ni-BTC MOF) for the borylation of aryl halides, including aryl chlorides, with bis(pinacolato)diboron, affording aryl boronate esters in high yields (up to >99% yield) with high selectivity. This protocol demonstrates broad functional group tolerance. Catalyst can be recyclable up to four times, and gram-scale reactions further highlights the usefulness of this method. In situ EPR experiments confirmed the formation of catalytically active Ni(I) species.

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase.

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Šresolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P21212 enabled the successful modeling of 96% of residues in the asymmetric unit, with final Rwork and Rfree values of 0.199 and 0.229, respectively.

Targeting mitochondrial shape: at the heart of cardioprotection.

There remains an unmet need to identify novel therapeutic strategies capable of protecting the myocardium against the detrimental effects of acute ischemia-reperfusion injury (IRI), to reduce myocardial infarct (MI) size and prevent the onset of heart failure (HF) following acute myocardial infarction (AMI). In this regard, perturbations in mitochondrial morphology with an imbalance in mitochondrial fusion and fission can disrupt mitochondrial metabolism, calcium homeostasis, and reactive oxygen species production, factors which are all known to be critical determinants of cardiomyocyte death following acute myocardial IRI. As such, therapeutic approaches directed at preserving the morphology and functionality of mitochondria may provide an important strategy for cardioprotection. In this article, we provide an overview of the alterations in mitochondrial morphology which occur in response to acute myocardial IRI, and highlight the emerging therapeutic strategies for targeting mitochondrial shape to preserve mitochondrial function which have the future therapeutic potential to improve health outcomes in patients presenting with AMI.

Deciphering the orthorhombic crystal structure of a novel NEIL1 nanobody with pseudo-merohedral twinning.

Nanobodies or VHHs (Variable Heavy domains of Heavy chain) are single domain antibodies that comprise three antigenic complementary determining regions (CDR). Nanobodies are used in numerous scientific applications including, bio-imaging, diagnosis, therapeutics, and macromolecular crystallography. We obtained crystals of a ∼14 kDa nanobody specific for the NEIL1 DNA glycosylase (hereafter called A5) in 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 1.0 M lithium sulfate monohydrate from the Crystal HT Hampton Research screen that were further optimized. Here, we describe the structure determination and refinement of the A5 crystals to a resolution of 2.1 Å. The data collected were complicated by the presence of anisotropy and twinning, and while initial space group determination pointed to a higher apparent tetragonal crystal system, the data statistics suggested twinning, placing the crystal in an orthorhombic system. Twinning was confirmed by the Padilla and Yeates test, H-test, and Britton test based on local intensity differences with a twin fraction of 0.4. Molecular replacement produced the best solution in the orthorhombic space group P2 1 2 1 2 with four molecules in the asymmetric unit and we were able to model over 96% of the residues in the electron density with a final R work and R free of 0.1988 and 0.2289 upon refinement. Synopsis: The crystal structure of a specific nanobody against NEIL1 was determined to 2.1 Å. The structure was ultimately solved in an orthorhombic space group after diffraction data analysis revealed mild anisotropy as well as pseudo-merohedral twinning.

Generation of an OCIAD2 overexpressing transgenic human embryonic stem cell line, BJNhem20-OCIAD2-OV.

Overexpression of Ovarian Carcinoma Immunoreactive Antigen Domain containing protein 2 (OCIAD2) was carried out in BJNhem20 human Embryonic Stem Cell line (hESC). A stable line was generated through nucleofection of the plasmid construct pCAG-OCIAD2.

Polß/XRCC1 heterodimerization dictates DNA damage recognition and basal Polß protein levels without interfering with mouse viability or fertility.

DNA Polymerase ß (Polß) performs two critical enzymatic steps during base excision repair (BER) - gap filling (nucleotidyl transferase activity) and gap tailoring (dRP lyase activity). X-ray repair cross complementing 1 (XRCC1) facilitates the recruitment of Polß to sites of DNA damage through an evolutionarily conserved Polß/XRCC1 interaction interface, the V303 loop. While previous work describes the importance of the Polß/XRCC1 interaction for human Polß protein stability and recruitment to sites of DNA damage, the impact of disrupting the Polß/XRCC1 interface on animal viability, physiology, and fertility is unknown. Here, we characterized the effect of disrupting Polß/XRCC1 heterodimerization in mice and mouse cells by complimentary approaches. First, we demonstrate, via laser micro-irradiation, that mouse Polß amino acid residues L301 and V303 are critical to facilitating Polß recruitment to sites of DNA damage. Next, we solved the crystal structures of mouse wild type Polß and a mutant protein harboring alterations in residues L301 and V303 (L301R/V303R). Our structural analyses suggest that Polß amino acid residue V303 plays a role in maintaining an interaction with the oxidized form of XRCC1. Finally, we created CRISPR/Cas9-modified Polb mice with homozygous L301R/V303R mutations (PolbL301R-V303R/L301R-V303R) that are fertile yet exhibit 15% reduced body weight at 17 weeks of age, as compared to heterozygous mice. Fibroblasts derived from PolbL301R-V303R/L301R-V303R mice demonstrate that mutation of mouse Polß's XRCC1 interaction domain leads to an ∼85% decrease in Polß protein levels. In all, these studies are consistent with a role for the oxidized form of XRCC1 in providing stability to the Polß protein through Polß/XRCC1 heterodimer formation.

Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A.

The maintenance of human mitochondrial DNA (mtDNA) is critical for proper cellular function as damage to mtDNA, if left unrepaired, can lead to a diverse array of pathologies. Of the pathways identified to participate in DNA repair within the mitochondria, base excision repair (BER) is the most extensively studied. Protein-protein interactions drive the step-by-step coordination required for the successful completion of this pathway and are important for crosstalk with other mitochondrial factors involved in genome maintenance. Human NEIL1 is one of seven DNA glycosylases that initiates BER in both the nuclear and mitochondrial compartments. In the current work, we scrutinized the interaction between NEIL1 and mitochondrial transcription factor A (TFAM), a protein that is essential for various aspects of mtDNA metabolism. We note, for the first time, that both the N- and C- terminal domains of NEIL1 interact with TFAM revealing a unique NEIL1 protein-binding interface. The interaction between the two proteins, as observed biochemically, appears to be transient and is most apparent at concentrations of low salt. The presence of DNA (or RNA) also positively influences the interaction between the two proteins, and molar mass estimates indicate that duplex DNA is required for complex formation at higher salt concentrations. Hydrogen deuterium exchange mass spectrometry data reveal that both proteins exchange less deuterium upon DNA binding, indicative of an interaction, and the addition of NEIL1 to the TFAM-DNA complex alters the interaction landscape. The transcriptional activity of TFAM appears to be independent of NEIL1 expression under normal cellular conditions, however, in the presence of DNA damage, we observe a significant reduction in the mRNA expression of TFAM-transcribed mitochondrial genes in the absence of NEIL1. Overall, our data indicate that the interaction between NEIL1 and TFAM can be modulated by local environment such as salt concentrations, protein availability, the presence of nucleic acids, as well as the presence of DNA damage.

PMS2 variant results in loss of ATPase activity without compromising mismatch repair.

Hereditary cancer syndromes account for approximately 5%-10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N-terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in-cell fluorescent-based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign.

Pan-RAS inhibitors: Hitting multiple RAS isozymes with one stone.

Mutations in the three RAS oncogenes are present in approximately 30% of all human cancers that drive tumor growth and metastasis by aberrant activation of RAS-mediated signaling. Despite the well-established role of RAS in tumorigenesis, past efforts to develop small molecule inhibitors have failed for various reasons leading many to consider RAS as "undruggable." Advances over the past decade with KRAS(G12C) mutation-specific inhibitors have culminated in the first FDA-approved RAS drug, sotorasib. However, the patient population that stands to benefit from KRAS(G12C) inhibitors is inherently limited to those patients harboring KRAS(G12C) mutations. Additionally, both intrinsic and acquired mechanisms of resistance have been reported that indicate allele-specificity may afford disadvantages. For example, the compensatory activation of uninhibited wild-type (WT) NRAS and HRAS isozymes can rescue cancer cells harboring KRAS(G12C) mutations from allele-specific inhibition or the occurrence of other mutations in KRAS. It is therefore prudent to consider alternative drug discovery strategies that may overcome these potential limitations. One such approach is pan-RAS inhibition, whereby all RAS isozymes co-expressed in the tumor cell population are targeted by a single inhibitor to block constitutively activated RAS regardless of the underlying mutation. This chapter provides a review of past and ongoing strategies to develop pan-RAS inhibitors in detail and seeks to outline the trajectory of this promising strategy of RAS inhibition.

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair.

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

Characterization of long G4-rich enhancer-associated genomic regions engaging in a novel loop:loop 'G4 Kissing' interaction.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

Extracellular vesicles - mediating and delivering cardioprotection in acute myocardial infarction and heart failure.

New treatments are urgently needed to reduce myocardial infarct size and prevent adverse post-infarct left ventricular remodeling, in order to preserve cardiac function, and prevent the onset of heart failure in patients presenting with acute myocardial infarction (AMI). In this regard, extracellular vesicles (EVs) have emerged as key mediators of cardioprotection. Endogenously produced EVs are known to play crucial roles in maintaining normal cardiac homeostasis and function, by acting as mediators of intercellular communication between different types of cardiac cells. Endogenous EVs have also been shown to contribute to innate cardioprotective strategies such as remote ischemic conditioning. In terms of EV-based therapeutics, stem cell-derived EVs have been shown to confer cardioprotection in a large number of small and large animal AMI models, and have the therapeutic potential to be applied in the clinical setting for the benefit of AMI patients, although several challenges need to be overcome. Finally, EVs may be used as vehicles to deliver therapeutics to the infarcted heart, providing a potential synergist approach to cardioprotection. In this review article, we highlight the various roles that EVs play as mediators and deliverers of cardioprotection, and discuss their therapeutic potential for improving clinical outcomes following AMI.

Stability and sub-cellular localization of DNA polymerase ß is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress.

Protein-protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein-protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase ß (Polß), we find that mutation of this surface threonine residue impacts critical Polß protein-protein interactions. We show that proteasome-mediated degradation of Polß is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein-protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polß in the cytosol via interaction between Polß and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polß with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polß to the nuclear compartment and regulates the stability of Polß via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polß/NQO1 complex, enhancing the interaction of Polß with XRCC1. Our results reveal that somatic mutations such as T304I in Polß impact critical protein-protein interactions, altering the stability and sub-cellular localization of Polß and providing mechanistic insight into how key protein-protein interactions regulate cellular responses to stress.

Mitochondrial DNA: Epigenetics and environment.

Maintenance of the mitochondrial genome is essential for proper cellular function. For this purpose, mitochondrial DNA (mtDNA) needs to be faithfully replicated, transcribed, translated, and repaired in the face of constant onslaught from endogenous and environmental agents. Although only 13 polypeptides are encoded within mtDNA, the mitochondrial proteome comprises over 1500 proteins that are encoded by nuclear genes and translocated to the mitochondria for the purpose of maintaining mitochondrial function. Regulation of mtDNA and mitochondrial proteins by epigenetic changes and post-translational modifications facilitate crosstalk between the nucleus and the mitochondria and ultimately lead to the maintenance of cellular health and homeostasis. DNA methyl transferases have been identified in the mitochondria implicating that methylation occurs within this organelle; however, the extent to which mtDNA is methylated has been debated for many years. Mechanisms of demethylation within this organelle have also been postulated, but the exact mechanisms and their outcomes is still an active area of research. Mitochondrial dysfunction in the form of altered gene expression and ATP production, resulting from epigenetic changes, can lead to various conditions including aging-related neurodegenerative disorders, altered metabolism, changes in circadian rhythm, and cancer. Here, we provide an overview of the epigenetic regulation of mtDNA via methylation, long and short noncoding RNAs, and post-translational modifications of nucleoid proteins (as mitochondria lack histones). We also highlight the influence of xenobiotics such as airborne environmental pollutants, contamination from heavy metals, and therapeutic drugs on mtDNA methylation. Environ. Mol. Mutagen., 60:668-682, 2019. © 2019 Wiley Periodicals, Inc.

Development of a Synthetic 3-ketosteroid Δ1-dehydrogenase for the Generation of a Novel Catabolic Pathway Enabling Cholesterol Degradation in Human Cells.

Cholesterol is an essential component of membranes, which is acquired by cells via receptor-mediated endocytosis of lipoproteins or via de novo synthesis. In specialized cells, anabolic enzymes metabolize cholesterol, generating steroid hormones or bile acids. However, surplus cholesterol cannot be catabolized due to the lack of enzymes capable of degrading the cholestane ring. The inability to degrade cholesterol becomes evident in the development and progression of cardiovascular disease, where the accumulation of cholesterol/cholesteryl-esters in macrophages can elicit a maladaptive immune response leading to the development and progression of atherosclerosis. The discovery of cholesterol catabolic pathways in Actinomycetes led us to the hypothesis that if enzymes enabling cholesterol catabolism could be genetically engineered and introduced into human cells, the atherosclerotic process may be prevented or reversed. Comparison of bacterial enzymes that degrade cholesterol to obtain carbon and generate energy with the action of human enzymes revealed that humans lack a 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), which catalyzes the C-1 and C-2 desaturation of ring A. Here we describe the construction, heterologous expression, and actions of a synthetic humanized Δ1-KstD expressed in Hep3B and U-937 cells, providing proof that one of three key enzymes required for cholesterol ring opening can be functionally expressed in human cells.

The Antitumor Drug LB-100 Is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating with the Active-Site Catalytic Metals in PPP5C.

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene.

Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss-of-function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%-30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42-44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small-angle X-ray scattering, and X-ray crystallography of the N-terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild-type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.