Unexpected exosites on the surface of matrix metalloproteinase-12 that fine-tune specificity for elastin and collagen V

Abstract only availableMMP-12 hydrolyzes collagens and elastin in normal physiological processes of development, wound repair, and reconstruction of the extracellular matrix. However, chronically elevated levels of MMP-12 activity can sustain disease states in certain tissues. Excessive MMP-12 activity upon elastic fibrils of lungs and arteries causes inflammation at those sites, resulting in emphysema, while excessive activity upon collagens triggers rupture of atheromatous plaques, which can lead to heart attack or stroke. Thus, research into the binding site interactions between MMP-12 and macromolecular substrates provides surface characterization of the enzyme and expedites the process of designing artificial inhibitor molecules to selectively inactivate MMP-12. The catalytic domain of MMP-12 spans amino acid residue 100 to 263, and this region is particularly important during hydrolysis of substrates. Three secondary binding sites within the catalytic domain were examined: phenylalanine 202 (F202), threonine 205 (T205), and histidine 206 (H206). Site-directed mutagenesis was performed at the aforementioned exosites to obtain single point mutations glutamine 202 (F202Q), lysine 205 (T205K), and histidine 206 (H206D). The MMP-12 mutants were expressed as the 18.2kDa catalytic domain in BL21 derived E. coli Rosetta™ 2 host strains. The mutated MMP-12 enzymes were isolated as insoluble inclusion bodies after lysis of E. coli cells using the French press. The inclusion bodies were solubilized in neutral 6M urea buffer, and preliminary purification with cation-exchange chromatography yielded a nucleic acid-free fraction of denatured enzyme. Refolding of the MMP-12 mutants was done by dialysis with serially diluted urea solution containing Tris-HCl buffer and CaCl2 at pH 7.5 without Zn++. ZnCl2 was added to the final dialysis buffer to complete the refolding process. Final purification of active renatured enzyme was achieved by another cation exchange chromatography run. Then, a Bio-Rad protein assay determined the concentration of the three MMP-12 mutants. Two types of flurometric-based kinetic experiments were performed to study changes in catalytic activity of the three MMP-12 mutants versus wild-type MMP-12: active site titration and substrate activity. Active site titration quantified the concentration of active sites for each MMP-12 mutant. The substrate runs for the substrates FS-6, fluorescently labeled elastin, and a triple helical peptide mimic of collagen V (THP-V) produced raw kinetics data, in the form of progress curves. Microcal Origin Pro 7.5 was used to analyze the data to give the kcat, the number of times each enzyme site converts substrate to product per second, and the Km, the concentration needed to achieve one half Vmax. Furthermore, dividing Kcat by Km revealed the amount of activity upon FS-6, elastin, and THP-V for each MMP-12 mutant with FS-6 as the control for mutated and wild-type MMP-12. Since the kcat/Km for wild-type MMP-12 was already known, comparison of activity upon THP-V and elastin between wild type MMP-12 and each MMP-12 mutant was achieved. Ongoing data collection suggests diminished catalytic activity upon elastin and THP-V in all three MMP-12 mutants (F202Q, T205K, and H206D) when compared to wild-type MMP-12, while showing no decrease in activity upon the general MMP substrate, FS-6. To date, data suggests that all three exosites are individually involved in fine-tuning MMP-12 specificity for elastin and triple helical peptide mimics of collagen V.NSF-REU Program in Biological Sciences & Biochemistr

Photoacoustic microscopy of human teeth

Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective

<i>Helicobacter pylori</i> extract induces purified neutrophils to produce reactive oxygen species only in the presence of plasma.

H. pylori is a bacterial pathogen infecting over half of the world's population and induces several gastric and extra-gastric diseases through its various virulence factors, especially cagA. These factors may be released from the bacteria during interactions with host immune cells. Neutrophils play key roles in innate immunity, and their activity is regulated by plasma factors, which can alter how these cells may interact with pathogens. The aim of the present study was to determine whether purified neutrophils could produce reactive oxygen species (ROS), one of the key functions of their anti-microbial functions, in response to extracts of cagA+ and cagA- H. pylori. Extracts from either cagA+ or cagA- H. pylori were co-cultured with human neutrophils in the presence or absence of plasma, and the neutrophil ROS production was measured. In the absence of plasma, extracts from cagA+ and cagA- H. pylori did not induce neutrophil ROS production, whereas in the presence of plasma, extracts from both cagA+ and cagA- H. pylori-induced ROS production. Furthermore, when peripheral blood mononuclear cells (PBMCs) were added to the purified neutrophils in the absence of plasma, there was no neutrophil ROS production after challenging with extracts from either cagA+ or cagA- H. pylori. Thus, it is suggested that plasma contains immunological components that change the responsiveness of neutrophils, such that when neutrophils encounter the bacterial antigens in H. pylori extracts, they become activated and produce ROS. This study also revealed a potential novel immunopathogenic pathway by which cagA activation of neutrophils contributed to inflammatory damage

Photoacoustic microscopy of human teeth

Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp