Expression, Characterization and Metallation Studies of Human Metallothionein Isoform 2a Using Electrospray Ionization Mass Spectrometry

Maintenance of the homeostasis of zinc (Zn) is very important in regulating bodily functions. There are over 300 Zn dependent enzymes identified, where Zn plays a structural or catalytic role. However, excess of Zn in a cell is toxic and free Zn ions are tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins, which can bind up to seven Zn ions and act as a Zn reservoir. The MT2a isoform is predominantly found in the liver. My research focused on the overexpression of human MT2a in Escherichia coli and the investigation of Zn binding pathways of MT2a in vitro. At physiological pH, Zn is terminally bound to the cysteine thiols of MT2a, making bead-like structures (non-cooperative metal binding), while at low pH, Zn formed clusters bridging the terminally bound Zn (cooperative metal binding). These findings draw our attention to investigate how other essential and toxic metals bind to MT2a. Keywords: Homeostasis, zinc, metallothionein (MT), metallothionein 2a (MT2a), non-cooperative, cooperative, Zn binding pathwa

Regulation of Metalloproteinase Activity is Critical for Pulmonary Microvascular Endothelial Cell Barrier Function

Sepsis is a life-threatening disease characterized by a dysregulated immune response to infection. Septic organ dysfunction is due to endothelial cell (EC) injury, including pulmonary microvascular ECs (PMVEC), resulting in loss of barrier function. Metalloproteinases, including matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAMs), and ADAMs with thrombospondin motifs (ADAMTS), may contribute to EC barrier dysfunction through cleavage of cell-cell junctions. The activity of metalloproteinases is regulated by tissue inhibitors of metalloproteinases (TIMPs). Although, previous studies suggest that metalloproteinases can cleave junctional proteins and thus disrupt the PMVEC barrier, the specific role of individual metalloproteinases in septic PMVEC barrier disruption is not known. I hypothesize that the inhibition of metalloproteinase activity by TIMPs supports normal EC barrier function. Moreover, loss of metalloproteinase inhibition leads to septic EC barrier dysfunction through inter-EC junctional protein degradation. Isolated mouse and human PMVEC were examined under basal (PBS) and septic (mouse: cytomix [equimolar tumour necrosis factor α, interferon γ, and interleukin 1β] + lipopolysaccharide [LPS]; human: cytomix) conditions. Gene expression was assessed using qRT-PCR (mouse) and RNA-Seq (human), and metalloproteinase activity was assessed using substrate-based assays. PMVEC permeability was quantified using Evans blue-labelled albumin, and surface localization of vascular endothelial (VE)-cadherin (adherens junction) and claudin 5 (tight junction) was assessed by immunofluorescence. Septic stimulation as well as the loss of TIMP3 led to disruption of inter-PMVEC junctional proteins and barrier dysfunction. ADAM17 activity was increased in the absence of TIMP3 and under septic conditions. Mmp13 expression and activity was increased in septic mouse PMVEC and the expression of ADAMTSs was increased in septic human PMVEC. Generally, treatment of septic mouse and human PMVEC as well as mouse PMVEC lacking TIMP3 with broad and selective metalloproteinase inhibitors reduced septic permeability and disruption of inter-PMVEC junctional proteins. Collectively, my findings indicate that the expression and activity of specific metalloproteinases mediates PMVEC barrier dysfunction and that TIMP3 supports barrier function through inhibition of metalloproteinases

Imbalance of Pulmonary Microvascular Endothelial Cell-Expression of Metalloproteinases and Their Endogenous Inhibitors Promotes Septic Barrier Dysfunction

Sepsis is a life-threatening disease characterized by excessive inflammation leading to organ dysfunction. During sepsis, pulmonary microvascular endothelial cells (PMVEC) lose barrier function associated with inter-PMVEC junction disruption. Matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases (ADAM), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), can cleave cell–cell junctional proteins, suggesting a role in PMVEC barrier dysfunction. We hypothesize that septic PMVEC barrier dysfunction is due to a disruption in the balance between PMVEC-specific metalloproteinases and TIMPs leading to increased metalloproteinase activity. The effects of sepsis on TIMPs and metalloproteinases were assessed ex vivo in PMVEC from healthy (sham) and septic (cecal ligation and perforation) mice, as well as in vitro in isolated PMVEC stimulated with cytomix, lipopolysaccharide (LPS), and cytomix + LPS vs. PBS. PMVEC had high basal Timp expression and lower metalloproteinase expression, and septic stimulation shifted expression in favour of metalloproteinases. Septic stimulation increased MMP13 and ADAM17 activity associated with a loss of inter-PMVEC junctional proteins and barrier dysfunction, which was rescued by treatment with metalloproteinase inhibitors. Collectively, our studies support a role for metalloproteinase–TIMP imbalance in septic PMVEC barrier dysfunction, and suggest that inhibition of specific metalloproteinases may be a therapeutic avenue for septic patients