Search tips
Search criteria

Results 1-12 (12)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Staphylococcus aureus sortase A contributes to the Trojan Horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation† 
Biochemistry  2011;50(35):7591-7599.
Staphylococcus aureus is a Gram-positive bacterial pathogen that causes serious infections which have become increasingly difficult to treat due to antimicrobial resistance and natural virulence strategies. Bacterial sortase enzymes are important virulence factors and good targets for future antibiotic development. It has recently been shown that sortase enzymes are integral to bacterial survival of phagocytosis, an underappreciated, but vital, step in S. aureus pathogenesis. Of note, the reaction mechanism of sortases relies on a solvent-accessible cysteine for transpeptidation. Due to the common strategy of oxidative damage employed by professional phagocytes to kill pathogens, it is possible that this cysteine may be oxidized inside the phagosome, thereby inhibiting the enzyme. This study addresses this apparent paradox by assessing the ability of physiological reactive oxygen species, hydrogen peroxide and hypochlorite, to inhibit sortase A (SrtA) from S. aureus. Surprisingly, we found that SrtA is highly resistant to oxidative inhibition, both in vitro and in vivo. The mechanism of resistance to oxidative damage is likely mediated by maintaining a high reduction potential of the catalytic cysteine residue, Cys184. This is due to the unusual active site utilized by S. aureus SrtA, which employs a reverse protonation mechanism for transpeptidation, resulting in a high pKa as well as reduction potential for Cys184. The results of this study suggest that S. aureus SrtA is able to withstand the extreme conditions encountered in the phagosome and maintain function, contributing to survival of phagocytotic killing.
PMCID: PMC3604699  PMID: 21812416
Sortase; Staphylococcus aureus; phagocytosis; oxidation; virulence; reverse protonation; reactive oxygen species
2.  Generation of Ramoplanin-Resistant Staphylococcus aureus 
FEMS microbiology letters  2010;310(2):104-111.
Ramoplanin is a lipoglycodepsipeptide antimicrobial active against clinically important Gram-positive bacteria including methicillin resistant Staphylococcus aureus. To proactively examine ramoplanin resistance, we subjected S. aureus NCTC 8325-4 to serial passage in the presence of increasing concentrations of ramoplanin, generating the markedly resistant strain RRSA16. Susceptibility testing of RRSA16 revealed the unanticipated acquisition of cross-resistance to vancomycin and nisin. RRSA16 displayed phenotypes, including a thickened cell wall and reduced susceptibility to Triton X-100 induced autolysis, which are associated with vancomycin intermediate resistant S. aureus strains. Passage of RRSA16 for 18 days in drug-free medium yielded strain R16-18d with restored antibiotic susceptibility. The RRSA16 isolate may be used to identify the genetic and biochemical basis for ramoplanin-resistance and further our understanding of the evolution of antibiotic cross-resistance mechanisms in S. aureus.
PMCID: PMC3596103  PMID: 20659164
ramoplanin; Staphylococcus aureus; vancomycin; VISA; nisin; cross-resistance
3.  Autocatalytic Intramolecular Isopeptide Bond Formation in Gram-Positive Bacterial Pili: A QM/MM Simulation 
Gram-positive pathogens possess external pili or fimbrae with which they adhere to host cells during the infection process. Unusual dual intramolecular isopeptide bonds between Asn and Lys side chains within the N-terminal and C-terminal domains of the pilus subunits have been observed initially in the Streptococcus pyogenes pilin subunit Spy0128 and subsequently in GBS52 from Streptococcus agalactiae, in the BcpA major pilin of Bacillus cereus and in the RrgB pilin of Streptococcus pneumoniae among others. Within each pilin subunit, intramolecular isopeptide bonds serve to stabilize the protein. These bonds provide a means to withstand large external mechanical forces, as well as possibly assisting in supporting a conformation favored for pilin subunit polymerization via sortase transpeptidases. Genome-wide analyses of pili-containing Gram-positive bacteria are known or suspected to contain isopeptide bonds in pilin subunits. For the autocatalytic formation of isopeptide crosslinks, a conservation of three amino acids including Asn, Lys, and a catalytically important acidic Glu (or Asp) residue are responsible. However, the chemical mechanism of how isopeptide bonds form within pilin remains poorly understood. Although it is possible that several mechanistic paths could lead to isopeptide bond formation in pili, the requirement of a conserved glutamate and highly organized positioning of residues within the hydrophobic environment of the active site were found in numerous pilin crystal structures such as Spy0128 and RrgB. This suggests a mechanism involving direct coupling of lysine side chain amine to the asparagine carboxamide mediated by critical acid/base or hydrogen bonding interactions with the catalytic glutamate residue. From this mechanistic perspective, we used the QM/MM minimum free-energy path method to examine the reaction details of forming the isopeptide bonds with the Spy0128 as a model pilin, specifically focusing on the role of the glutamate in catalysis. It was determined that the reaction mechanism likely consists of two major steps: the nucleophilic attack on Cγ by nitrogen in the unprotonated Lys ε-amino group and then two concerted proton transfers occur during the formation of the intramolecular isopeptide bond to subsequently release ammonia. More importantly, within the dual active sites of Spy0128, Glu117 and Glu258 residues function as crucial catalysts for each isopeptide bond formation, respectively, by relaying two proton transfers. This study also suggests that domain-domain interactions within Spy0128 may modulate the reactivity of residues within each active site. Our results may hopefully shed light on the molecular mechanisms of pilin biogenesis in Gram-positive bacteria.
PMCID: PMC3081525  PMID: 21142157
Pilus; pili; Gram-positive bacteria; isopeptide bond; QM/MM-MFEP; reaction path optimization; QM/MM simulation
4.  Monitoring β-Secretase Activity in Living Cells with a Membrane Anchored FRET Probe** 
PMCID: PMC3495557  PMID: 23023944
Alzheimer’s Disease; β-Secretase; FRET; Imaging agents; Analytical Methods
5.  Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein 
Macromolecular rapid communications  2013;34(15):1256-1260.
Conventional methods for synthesizing protein/peptide–polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site-specificity, which results in heterogeneous products with significantly compromised bio activity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with > 90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide–polymer conjugates.
PMCID: PMC3754797  PMID: 23836349
atom transfer radical polymerization (ATRP); biological application of polymers; grafting from; protein–polymer conjugates; sortase
6.  Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and independent manners 
ACS chemical biology  2012;7(7):1221-1231.
Lysine specific demethylase 1 (LSD1, also known as KDM1) is a histone modifying enzyme that regulates the expression of many genes important in cancer progression and proliferation. It is present in various transcriptional complexes including those containing the estrogen receptor (ER). Indeed, inhibition of LSD1 activity and or expression has been shown to attenuate estrogen signaling in breast cancer cells in vitro implicating this protein in the pathogenesis of cancer. Herein we describe experiments that utilize small molecule inhibitors, phenylcyclopropylamines, along with small interfering RNA to probe the role of LSD1 in breast cancer proliferation and in estrogen-dependent gene transcription. Surprisingly, whereas we have confirmed that inhibition of LSD1 strongly inhibits proliferation of breast cancer cells, we have determined that the cytostatic actions of LSD1 inhibition are not impacted by ER-status. These data suggest that LSD1 may be a useful therapeutic target in several types of breast cancer; most notably inhibitors of LSD1 may have utility in the treatment of ER-negative cancers for which there are minimal therapeutic options.
PMCID: PMC3582702  PMID: 22533360
Lysine demethylase; phenylcyclopropylamines; estrogen receptor; breast cancer
7.  Chlamydia Protease-Like Activity Factor (CPAF): Characterization of Proteolysis Activity In Vitro and Development of a Nanomolar Affinity CPAF Zymogen-Derived Inhibitor 
Biochemistry  2011;50(35):7441-7443.
During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease CPAF into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Here we report the development of an in vitro assay for the enzyme and the discovery of a cell-permeable CPAF zymogen-based peptide inhibitor with nanomolar inhibitory affinity. Treating C. trachomatis-infected HeLa cells with this inhibitor prevented CPAF cleavage of the intermediate filament vimentin, and led to loss of vimentin-cage surrounding the intracellular vacuole. Because Chlamydia is a genetically intractable organism, this inhibitor may serve as a tool to understand the role of CPAF in pathogenesis.
PMCID: PMC3608462  PMID: 21830778
Chlamydia trachomatis; inhibitor; pathogenesis; protease
8.  Thermodynamic Characterization of the Binding Interaction between the Histone Demethylase LSD1/KDM1 and CoREST 
Biochemistry  2010;50(4):546-557.
Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, the histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDACs 1/2) and the scaffolding protein CoREST. Although little is known concerning the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST286–482 bound to an alpha helical coiled-coil tower domain within LSD1 has been recently reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means to inhibit LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Towards this end, isothermal titration calorimetry studies (ITC) were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST286–482. The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (Kd) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant pressure heat capacity change (ΔCp) of the interaction was determined to be −0.80 ± 0.01 kcal/mol·K. Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within residues 293–380, also known as the CoREST ‘linker’ region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region293–380 of CoREST are similar to those obtained from the interaction between LSD1 and CoREST286–482. These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.
PMCID: PMC3604701  PMID: 21142040
histone demethylase; flavoenzyme; amine oxidase; LSD1; CoREST; isothermal titration calorimetry (ITC); protein-protein interactions
9.  Use of pH and Kinetic Isotope Effects to Establish Chemistry As Rate-Limiting in Oxidation of a Peptide Substrate by LSD1† 
Biochemistry  2009;48(23):5440-5445.
The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the N-terminal 21 residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, kred, equals kcat, establishing the reductive half reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 ± 0.2 on the kred, kcat and kcat/Km values for the peptide, establishing CH bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin are detectable by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The kcat/Km value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The D(kcat/Km) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.
PMCID: PMC2738601  PMID: 19408960
10.  Probing of the cis-5-Phenyl Proline Scaffold as a Platform for the Synthesis of Mechanism-Based Inhibitors of the Staphylococcus aureus Sortase SrtA Isoform 
Bioorganic & medicinal chemistry  2009;17(7):2886-2893.
cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit S. aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.
PMCID: PMC2663005  PMID: 19269184
S. aureus; Sortase; Inhibitor; Vinyl Sulfone
11.  Critical Role of NOD2 in Regulating the Immune Response to Staphylococcus aureus▿  
Infection and Immunity  2009;77(4):1376-1382.
NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2−/−) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (107 bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2−/− and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2−/− mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2−/− and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2−/− mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2−/− mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1β, IL-4, IL-6, and IL-10) were similar in Nod2−/− and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.
PMCID: PMC2663139  PMID: 19139201
12.  Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B 
A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.
PMCID: PMC2661354  PMID: 18242989

Results 1-12 (12)