Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: Interplay between epigenetics and periodontal infection

ObjectivePeriodontal pathogens initiate chronic dysregulation of inflammation and tissue homeostasis that characterize periodontal disease. To better understand oral microbe-host tissue interactions, we investigated expression and activation of MMP-2 in periodontal ligament cells following Treponema denticola challenge.DesignCultured PDL cells were challenged with T. denticola, and bacterial adherence, internalization and survival were assayed by immunofluorescence microscopy and antibiotic protection assays, respectively. MMP-2 activation was detected by zymography. MMP-2, MT1/MMP and TIMP-2 expression following T. denticola challenge was determined by qRT-PCR. Promoter methylation of MMP-2 and MT1/MMP was screened by methylation-sensitive restriction analysis and by bisulfite DNA sequencing.ResultsT. denticola adhered to and was internalized by PDL cells but did not survive intracellularly beyond 24h. Importantly, while dentilisin activity in PDL culture supernatants gradually decreased following T. denticola challenge, MMP-2 activation persisted for up to 5 days, suggesting involvement of other regulatory mechanisms. Transcription and expression of MT1/MMP and TIMP-2 increased in response to T. denticola challenge. However, consistent with previously reported constitutive pro-MMP-2 expression in PDL cells, the MMP-2 promoter was hypomethylated, independent of T. denticola challenge.ConclusionsMMP-2 promoter hypomethylation is consistent with constitutive pro-MMP-2 expression in PDL cells. This, coupled with T. denticola-mediated upregulation of MMP-2-related genes and chronic activation of pro-MMP-2, mimics key in vivo mechanisms of periodontal disease chronicity, in particular MMP-2-dependent matrix degradation and bone resorption. Adherence and/or internalization of T. denticola may contribute to these processes by one or more regulatory mechanisms, including contact-dependent signal transduction or other epigenetic mechanisms

Calsyntenin-3 molecular architecture and interaction with neurexin 1α.

Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca(2+), and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca(2+) and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function

Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2).

Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule