Treponema denticola increases MMP‐2 expression and activation in the periodontium via reversible DNA and histone modifications

Host‐derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP‐2 expression and activation in periodontal ligament (PDL) cells, and dentilisin‐mediated activation of pro‐MMP‐2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP‐2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP‐2 expression, and activation were assessed by immunoblot, zymography, and qRT‐PCR, respectively. Chromatin modification enzyme expression in T. denticola‐challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola‐challenged PDL cells. T. denticola‐mediated MMP‐2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola‐mediated MMP‐2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142926/1/cmi12815.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142926/2/cmi12815_am.pd

Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp)

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKS IE GDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G–D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G–D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein. © 2011 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78496/1/202_ftp.pd

Affinity Labeling of RNA Polymerase II in the Transcriptionally Active Complex by a Phosphorylating Analog of the Initiation Substrate

RNA polymerase II is composed of 12 subunits Substrate derivatives with various chemical groups were used to study the active site of RNA polymerase II. Reagents with arylazide groups were activated by irradi ation at the appropriate wavelength. An additional reagent was required for detection of the products of pro tein interaction with other reagents. For example, when carbonyl containing reagents were used for irreversible attachment of an affinity reagent to the protein, the gen erated Schiff bases were reduced with NaBH 4 . These studies were performed with highly purified enzyme preparations , poly[d(A T)] ACCELERATED PUBLICATION 0006 2979/00/6510 1129$25.00 ©2000 MAIK "Nauka / Interperiodica" * To whom correspondence should be addressed. Vol. 65, No. 10, 2000, pp. 1129 1134. Translated from Biokhimiya, Vol. 65, No. 10, 2000, pp. 1334 1340. Original Russian Text Copyright © 2000 Abstract-Affinity modification of RNA polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5′ terminal phosphate group with a 4 N,N dimethylaminopyridine residue (DMAP pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA polymerase II from modification. The third subunit may be involved in the formation of the substrate binding site of the enzyme