Showing papers by "Robert McKenna published in 2013"
TL;DR: The favourable properties of hCA II such as fast kinetics, easy expression and purification, high solubility and intermediate heat resistance have made it an attractive candidate for numerous industrial applications.
Abstract: Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3−. Of the 15 isoforms of human (h) α-CA, 12 are catalytic (hCAs I-IV, VA, VB, VI, VII, IX, XII-XIV). The remaining three acatalytic isoforms (hCAs VIII, X and XI) lack the active site Zn2+ and are referred to as CA-related proteins (CA-RPs); however, their function remains elusive. Overall these isoforms are very similar to each other in structure but they differ in their expression and distribution. The favourable properties of hCA II such as fast kinetics, easy expression and purification, high solubility and intermediate heat resistance have made it an attractive candidate for numerous industrial applications. This review highlights the structural similarity and stability comparison among hCAs.
184 citations
TL;DR: This review focuses on the mechanism of epilepsy, CA catalysis, and recent developments in the treatment of epilepsy using CAIs.
Abstract: Introduction: An epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. The International League Against Epilepsy classifies seizures in two broad categories: partial (localized to one cerebral hemisphere) and generalized (localized to both cerebral hemispheres). One indirect pathway for the treatment of epilepsy includes the inhibition of carbonic anhydrase (CA), thereby increasing CO2 levels in the brain. Areas covered: Carbonic anhydrases (EC 4.2.1.1) are ubiquitous metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3 -, respectively. CA inhibitors (CAIs) such as acetazolamide, methazolamide, topiramate, zonisamide, and sulthiame can reduce seizures through perturbation of the CO2 equilibrium and/or the inhibition of ion channels. This review focuses on the mechanism of epilepsy, CA catalysis, and recent developments in the treatment of epilepsy using CAIs. Expert opinion: Based on the observ...
173 citations
TL;DR: This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus.
Abstract: The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus.
136 citations
TL;DR: Serotypes of adeno-associated virus (AAV), AAV1, AAV2, A AV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability to provide direction for future studies on entry and vector production.
Abstract: Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.
106 citations
TL;DR: The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.
Abstract: Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.
105 citations
TL;DR: This article focuses on CA IX inhibition and two different structural approaches to CA isoform specific drug designing: tail approach and fragment addition approach.
102 citations
TL;DR: The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity.
85 citations
TL;DR: A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities.
Abstract: The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.
67 citations
TL;DR: This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.
Abstract: Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 μs–1 for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 μs–1 for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the ...
55 citations
TL;DR: Carbonic anhydrases (CAs) are among the fastest enzymes known, which have a maximum catalytic efficiency approaching the diffusion limit of 108m−1s−1.
Abstract: As the global atmospheric emissions of carbon dioxide (CO2) and other greenhouse gases continue to grow to record-setting levels, so do the demands for an efficient and inexpensive carbon sequestration system. Concurrently, the first-world dependence on crude oil and natural gas provokes concerns for long-term availability and emphasizes the need for alternative fuel sources. At the forefront of both of these research areas are a family of enzymes known as the carbonic anhydrases (CAs), which reversibly catalyze the hydration of CO2 into bicarbonate. CAs are among the fastest enzymes known, which have a maximum catalytic efficiency approaching the diffusion limit of 108 M−1s−1. As such, CAs are being utilized in various industrial and research settings to help lower CO2 atmospheric emissions and promote biofuel production. This review will highlight some of the recent accomplishments in these areas along with a discussion on their current limitations.
43 citations
TL;DR: It is shown that SFMBT1 is essential for Snai1-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGFβ.
TL;DR: Kinetic studies utilizing the measurement of (18)O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII.
Abstract: The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 A resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of 18O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.
19 Aug 2013
TL;DR: The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others.
Abstract: The carbonic anhydrases (CAs) are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II) has a catalytic efficiency of 108 M−1 s−1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development.
TL;DR: Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice showed both similarity in structure and analogous interactions, which suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation.
Abstract: The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-A resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors.
TL;DR: The in vitro action of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is characterized to provide a basis for future research projects to develop the newly synthesized compound into a clinically usable anticancer agent either alone or in combination with other agents.
Abstract: This research was supported by grants from the Medical Research Council of South Africa (AG374, AK076), the Cancer Association of South Africa (AK246), National Research Foundation (NRF) (AL239), the Research Committee of the Faculty of Health Sciences (RESCOM) of University of Pretoria and the
Struwig-Germeshuysen Cancer Research Trust of South Africa (AJ038, AN074).
TL;DR: Videothoracoscopy restaging was "feasible" in this prospective multi-institutional trial and provided pathologic specimens of the ipsilateral nodes and was limited by radiation and the 4R nodal station.
TL;DR: Sulfonamide was a medium potency hCA I and II inhibitor but was highly effective as a hCA IX and XII inhibitor, which has been explained by resolving the crystal structure, and may be used to design more isoform-selective compounds.
TL;DR: Important differences of binding emerged, which explain the highly different inhibition profile of the two compounds against the investigated CA isoforms, most of which (CA I, II, IX and XII) are important drug targets.
TL;DR: The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis.
TL;DR: The seventh edition of the AJCC/UICC lung cancer staging system appears to be an improvement over the preceding system and survival analysis and Kaplan-Meier survival curves showed more monotonic progression, distinction and homogeneity within groups in the seventh edition.
Abstract: Objectives A new revision of the international lung cancer staging system has been recently introduced. The revisions are largely focussed on the T descriptor. We sought to test the validity of this new system on a separate prospectively collected cohort of patients from a recent multicentre trial of early-stage lung cancer. Methods We reviewed the prospectively collected data from 1012 patients undergoing pulmonary resection for early-stage lung cancer in the ACOSOG Z0030 trial. TNM descriptors and overall staging were assessed using both the sixth and seventh editions of the American Joint Committee on Cancer and the Union Internationale Contre le Cancer (AJCC/UICC) lung cancer staging system. Survival results were analysed according to both staging allocations. Results Using the proposed criteria, the number of patients by stage in the sixth and seventh edition allocations, respectively, were as follows: IA (432, 431); IB (402, 303); IIA (39, 167); IIB (94, 70); IIIA (26, 40); IIIB (19,0); there were no stage IV patients by either version. Overall, 180 (18%) patients had a change in the stage group from the sixth to seventh edition versions with 76 (8%) being downstaged and 104 (10%) being upstaged. In the sixth edition staging system based on pathological stages, median survivals in years were as follows: IA, NA; IB, 7.7; IIA, 4.0; IIB, 3.6; IIIA, 2.6 and IIIB, 2.4. Five-year survivals were: IA, 76.4%; IB, 62.0%; IIA, 47.8%; IIB, 40.4%; IIIA, 31.3% and IIIB, 44.4%. In the new system, median survivals in years were as follows: IA, NA; IB, 8.2; IIA, 4.4; IIB, 3.6 and IIIA, 1.8. Five-year survivals were: IA, 76.9%; IB, 65.0%; IIA, 48.5%; IIB, 42.9% and IIIA, 30.6%. Survival analysis and Kaplan-Meier survival curves showed more monotonic progression, distinction and homogeneity within groups in the seventh edition. Conclusions This study provides an external validation of the recently revised lung cancer staging system using a large multicentre database. The seventh edition of the AJCC/UICC lung cancer staging system appears to be an improvement over the preceding system.
TL;DR: Results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability ofCA II nor the binding of AZM in the crystal structure or in solution, however, addition of GOL does affect the kinetics of CA II.
Abstract: Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.
TL;DR: The main conclusion of the study is that the orientation of the tail moiety and the presence of flexible linkers as well polar groups in it, strongly influence the potency and the selectivity of the sulfonamides for the inhibition of cytosolic, mitochondrial or transmembrane CA isoforms.
TL;DR: X-ray crystallographic analysis suggests that the structural basis of this destabilization is via disruption and/or removal of weak C-H⋯π interactions between F226 to F66, F95 and W97.
TL;DR: It is shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner, and the dynamic phosphorylation status of H5 influences this structure and has implications for the role of H 5 in multiple processes during virus replication.
TL;DR: Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials, and three genes, pa0102, pa2053 and pa4676, encoding three functional β-carbonic anhydrases could serve as novel antimicrobial drug targets for this pathogen.
Abstract: Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional β-carbonic anhydrases (β-CAs): psCA1, psCA2 and psCA3, respectively. These β-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of β-CA psCA3. Form A diffracted to a resolution of 2.9 A; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 A, and had a calculated Matthews coefficient of 2.23 A3 Da−1 assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 A; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 A, and had a calculated Matthews coefficient of 2.48 A3 Da−1 assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.
TL;DR: A recent study as discussed by the authors showed that the 1.1 Å resolution Neutron protein structure directly showed 95% of all H atoms, compared to only 30% in the 0.85Å resolution X-ray structure.
Abstract: Volume 24 • Number 4 • 2013 Neutron News 20 Background Hydrogens (H) in structural biology – detection and function About half the atoms in proteins consist of hydrogen (H), with the other half being a combination of mostly C, N, O, and some S. In addition to these lighter atoms, metalloproteins also contain small amounts of bioactive metals, such as Mg, Ca, Zn, Mn, Fe, and a few others. Despite being the most abundant atom in proteins, H is the most diffi cult to observe in conventional X-ray crystal structures, due to their very small X-ray scattering length. H atoms are very important to protein folding, hydrogen bonding interactions, substrate or inhibitor binding, enzyme catalysis, and protein solvation [1]. Enzymes can also use H2O, OH–, H+, and H3O + to mediate catalysis but distinguishing between these species is very challenging using traditional methods [2, 3]. H is typically only defi nitively observed in X-ray structures at ultra high resolution (better than 1 Å) and even then, only a fraction of the total is observed. Seeing as ultra high-resolution structures only comprise ~0.7% of total deposited structures in the PDB (Protein Data Bank http://www.pdb.org), there is very little direct experimental information on H atom positions in proteins. Highlighting this, a recent study of the highest resolution neutron protein structure ever determined showed that the 1.1 Å resolution neutron structure directly showed ~95% of all H atoms, compared to only 30% in the 0.85 Å resolution X-ray structure [1]. Neutron protein crystallography offers unique information on H atoms and is highly complementary to X-ray scattering. From the atom types found in proteins, neutrons are scattered to a similar extent and offers isotope discrimination between H and deuterium (D), each with scattering lengths of –3.7 and 6.7 fm, respectively. To overcome signal cancellation and the large incoherent background from H scattering, it is routine to exchange all possible H atoms for its isotope D for neutron diffraction. This facilitates the direct observation of the protonation state of amino acid side chains, water orientation, H-bonding, and ligand binding [1, 2].
TL;DR: The studies and evidence in support of the potential benefits afforded by the video-assisted thoracic surgery approach in the treatment of lung cancer are reviewed.
Abstract: SUMMARY Anatomic lung resection remains the gold standard in the treatment of lung cancer. The traditional approach has been an open thoracotomy with anatomic lobectomy. The approach to the operation has continued to evolve, transitioning from large thoracotomy incisions to smaller muscle sparing incisions to video-assisted thoracic surgery. This article reviews the studies and evidence in support of the potential benefits afforded by the video-assisted thoracic surgery approach in the treatment of lung cancer.