Mechanistic investigations of matrix metalloproteinase-8 inhibition by metal abstraction peptide.

The mechanism of matrix metalloproteinase-8 (MMP-8) inhibition was investigated using ellipsometric measurements of the interaction of MMP-8 with a surface bound peptide inhibitor, tether-metal abstraction peptide (MAP), bound to self-assembled monolayer films. MMP-8 is a collagenase whose activity and dysregulation have been implicated in a number of disease states, including cancer metastasis, diabetic neuropathy, and degradation of biomedical reconstructions, including dental restorations. Regulation of activity of MMP-8 and other matrix metalloproteinases is thus a significant, but challenging, therapeutic target.

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5.

Probing the dual function of a novel tertiary amine compound in dentin adhesive formulations.

Objectives: A novel tertiary amine compound containing three methacrylate-urethane groups was synthesized for application in dentin adhesives. The synthesis, photopolymerization kinetics, and leaching were examined in an earlier study using this novel compound as the co-initiator (0.5 and 1.

Cytochrome P450 17A1 Interactions with the FMN Domain of Its Reductase as Characterized by NMR.

To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein.

Effect of crosslinking density of polymers and chemical structure of amine-containing monomers on the neutralization capacity of dentin adhesives.

Objectives: Neutralization of the acidic micro-environment at the tooth/material interface is expected to provide enhanced durability for dental composite restorations. The objective of this study is to explore the effect of amine-containing monomer formulations and the crosslinking density of the resultant polymers on the neutralization capacity.

Materials And Methods: The co-monomer system was varied systematically to obtain different proportions of Bisphenol A glycerolate dimethacrylate (BisGMA) and 2-hydroxyethyl methacrylate (HEMA), while maintaining a constant amount of amine-containing methacrylate monomer.

Do ASARM peptides play a role in nephrogenic systemic fibrosis?

Nephrogenic systemic fibrosis (NSF) is a devastating condition associated with gadolinium (Gd3+)-based contrast agents (GBCAs) in patients with kidney disease. The release of toxic Gd3+ from GBCAs likely plays a major role in NSF pathophysiology. The cause and etiology of Gd3+ release from GBCAs is unknown.

Metal ion interactions with mAbs: Part 1.

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu(2+), can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu(2+)-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu(2+).

Synthesis and evaluation of a novel co-initiator for dentin adhesives: polymerization kinetics and leachables study.

A new tertiary amine co-initiator (TUMA) containing three methacrylate-urethane groups was synthesized for application in dentin adhesives. The photopolymerization kinetics and leaching of unreacted components from methacrylate-based dental polymers formulated with this new co-initiator were determined. The newly synthesize co-initiator showed good chemical stability and decreased amine release from the initiator system.

Development of methacrylate/silorane hybrid monomer system: Relationship between photopolymerization behavior and dynamic mechanical properties.

Resin chemistries for dental composite are evolving as noted by the introduction of silorane-based composites in 2007. This shift in the landscape from methacrylate-based composites has fueled the quest for versatile methacrylate-silorane adhesives. The objective of this study was to evaluate the polymerization behavior and structure/property relationships of methacrylate-silorane hybrid systems.

Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.

The transcription factor atonal homolog 1 (ATOH1) has multiple homologues that are functionally conserved across species and is responsible for the generation of sensory hair cells. To evaluate potential functional differences between homologues, human and mouse ATOH1 (HATH1 and MATH-1, respectively) were nonvirally delivered to human Wharton's jelly cells (hWJCs) for the first time. Delivery of HATH1 to hWJCs demonstrated superior expression of inner ear hair cell markers and characteristics than delivery of MATH-1.

Characterization of Acid-neutralizing Basic Monomers in Co-solvent Systems by NMR.

Metabolic activity of the oral microbiota leads to acidification of the microenvironment and promotes demineralization of tooth structure at the margin of composite restorations. The pathogenic impact of the biofilm at the margin of the composite restoration could be reduced by engineering novel dentin adhesives that neutralize the acidic micro-environment. Integrating basic moieties into methacrylate derivatives has the potential to buffer against acid-induced degradation, and we are investigating basic monomers for this purpose.

Stability analysis of an inline peptide-based conjugate for metal delivery: nickel(II)-claMP Tag epidermal growth factor as a model system.

Metals are a key component of many diagnostic imaging and biotechnology applications, and the majority of cancer patients receive a platinum-based drug as part of their treatment. Significant effort has been devoted to developing tight binding synthetic chelators to enable effective targeted delivery of metal-based conjugates, with most successes involving lanthanides rather than transition metals for diagnostic imaging. Chemical conjugation modifies the protein's properties and generates a heterogeneous mixture of products.

Grafting MAP peptide to dental polymer inhibits MMP-8 activity.

Matrix metalloproteinases (MMPs) are a class of zinc and calcium-dependent endopeptidases responsible for degrading extracellular matrix (ECM) components. Their activity is critical for both normal biological function and pathological processes (Dejonckheere et al., Cytokine Growth Factor Rev 2011;22:73-81).

claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals.

Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1).

Synthesis and evaluation of novel dental monomer with branched carboxyl acid group.

To enhance the water miscibility and increase the mechanical properties of dentin adhesives, a new glycerol-based monomer with vinyl and carboxylic acid, 4-((1,3-bis(methacryloyloxy)propan-2-yl)oxy)-2-methylene-4-oxobutanoic acid (BMPMOB), was synthesized and characterized. Dentin adhesive formulations containing 2-hydroxyethyl methacrylate (HEMA), 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyl]propane (BisGMA), and BMPMOB were characterized with regard to real-time photopolymerization behavior, water sorption, dynamic mechanical analysis, and microscale three-dimensional internal morphologies and compared with HEMA/BisGMA controls. The experimental adhesive copolymers showed higher glass transition temperature and rubbery moduli, as well as improved water miscibility compared to the controls.

(1)H, (13)C and (15)N backbone assignment of the EC-1 domain of human E-cadherin.

The Extracellular 1 (EC1) domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e.

Multivariate analysis of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopic data to confirm phase partitioning in methacrylate-based dentin adhesive.

Water is ubiquitous in the mouths of healthy individuals and is a major interfering factor in the development of a durable seal between the tooth and composite restoration. Water leads to the formation of a variety of defects in dentin adhesives; these defects undermine the tooth-composite bond. Our group recently analyzed phase partitioning of dentin adhesives using high-performance liquid chromatography (HPLC).

Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges.

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known.

Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates.

Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics, and tissue distributions.

Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5.

Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters the specificity of the reaction pathway.

The unique metal abstracting peptide asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to dld-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion.

Effect of pH and excipients on structure, dynamics, and long-term stability of a model IgG1 monoclonal antibody upon freeze-drying.

Purpose: To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.

Methods: IgG1 mAb was formulated with mannitol at pH 3.0, 5.

Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway.

Controlling the chiral inversion reaction of the metallopeptide Ni-asparagine-cysteine-cysteine with dioxygen.

Synthetically generated metallopeptides have the potential to serve a variety of roles in biotechnology applications, but the use of such systems is often hampered by the inability to control secondary reactions. We have previously reported that the Ni(II) complex of the tripeptide LLL-asparagine-cysteine-cysteine, LLL-Ni(II)-NCC, undergoes metal-facilitated chiral inversion to dld-Ni(II)-NCC, which increases the observed superoxide scavenging activity. However, the mechanism for this process remained unexplored.