Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Gerard O’Cuinn in Metabolism of Brain Peptides, 2020
This assay served as a convenient means for the purification of the responsible activity. Inactive proteins were removed by acid precipitation, and the enzyme further purified by chromatography on columns of DEAE Cellulose, Sephadex G-100, and phenyl Sepharose®. A 300 fold purified enzyme was characterized having a pH optimum of 7.0 and a molecular weight of 67,000. The enzyme was weakly inhibited by thiol blocking reagents. Dithiothreitol had a dual effect, stimulating at low concentrations and inhibiting at high concentrations. Metal chelators such an EDTA and o-phenanthroline were inhibitory. Dialysis against EDTA abolished activity, and activity could be restored by the addition of Zn++. The enzyme was therefore classified as a metalloproteinase, a classification verified by subsequent cloning. After having been assigned an E.C. number, the enzyme was subsequently referred to by Orlowski and co-workers as “endopeptidase 24.15”.
Tissue Responses to Infection and Injury
Jeremy R. Jass in Understanding Pathology, 2020
Collagen is synthesised in the form of three alpha chains entwined in a helix. A key step in this process is the hydroxylation of the amino acid proline requiring the presence of vitamin C. Vitamin C deficiency will therefore delay wound healing. Other factors inhibiting wound healing include mechanical interference, infection, foreign material and steroid use. The procollagen is then crosslinked (outside the fibroblast) to form collagen fibrils. Tensile strength within a wound is a factor of both the amount and structural orientation of the collagen fibres. Remodelling is an active process involving both synthesis and controlled degradation of collagen. Degradation is achieved by a family of zinc-dependent enzymes called metalloproteinases, which are synthesised by fibroblasts, inflammatory cells and some epithelial cells. Metalloproteinase activity is controlled by a further family of enzymes called tissue inhibitors of metalloproteinase (TIMP). Expression of metalloproteinases is thought to be a key step in the early invasion of cancer, which involves the degradation of extracellular matrix proteins. A cancer may therefore be described as a wound that never heals.
Articular Cartilage
Manoj Ramachandran, Tom Nunn in Basic Orthopaedic Sciences, 2018
The matrix metalloproteinases are classified into collagenases, stromelysins, gelatinases and membrane-associated metalloproteinases. These degrade collagen and proteoglycan aggregates as part of the normal turnover of the matrix constituents. Balancing this action are the proteinase inhibitors. Tissue inhibitors of matrix metalloproteinase (TIMPs) are acidic polypeptides that prevent degradation by the metalloproteinases by binding to the matrix proteins. The avascular nature of articular cartilage is maintained by TIMPs that inhibit the proteases produced by migrating vascular endothelium. TIMPs may form the basis of future drugs that inhibit the excessive expression of matrix metalloproteinases, as this may be responsible for the progression and severity of osteoarthritis.
Involvement of NF-κB signaling pathway in the regulation of PRKAA1-mediated tumorigenesis in gastric cancer
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Yangmei Zhang, Xichang Zhou, Qinglin Zhang, Youwei Zhang, Xiang Wang, Long Cheng
Matrix metalloproteinase is an important factor in the degradation and remodeling of the extracellular matrix. MMP-2 is an important member of the family, regulated by NF-κB signaling and closely related to the occurrence and metastasis of malignant tumors [43,44]. Inhibiting NF-κB signaling pathway significantly abolished the cell invasion and up-regulation of MMP-2 in GC cells [45]. Our study showed that PRKAA1 knockdown significantly inhibited NF-κB activation and MMP-2 expression. These data suggest that PRKAA1 may regulate cell migration and invasion through NF-κB/MMP2 signaling pathway. In line with our findings that PRKAA1 knockdown significantly attenuated AICAR-induced NF-κB activation in human leukemic cell line THP-1 [46]. Inhibiting NF-κB signaling and expression of MMP2 markedly suppressed the invasion and migration of B16F-10 melanoma cells and AGS gastric adenocarcinoma cells in vitro [47,48]. Our rescue experiments found that PRKAA1 overexpression significantly inhibited NF-κBp50 knockdown-mediated the decrease of GC cell invasion and migration while PRKAA1 knockdown significantly inhibited NF-κBp50 overexpression-induced the increase of GC cell invasion and migration, which indicated that PRKAA1 may associate with NF-κBp50-induced the invasion and migration of GC cells.
Thermodynamic profiling for fragment-based lead discovery and optimization
Published in Expert Opinion on Drug Discovery, 2020
György G. Ferenczy, György M. Keserű
One of the first studies on the impact of binding thermodynamics on fragment optimization has been published by Bertini et al. [93]. The authors investigated the optimization of low affinity fragments to nanomolar MMP12 inhibitors by evaluating the key compounds along the optimization path by biochemical and ITC measurements, and X-ray crystallography. Matrix metalloproteinases are zinc containing proteases. Most of the high affinity MMP inhibitors form direct interactions with the zinc ion located at the substrate binding groove. Due to the chelating properties of hydroxamic acids, the identification of the low affinity and nonspecific acetohydroxamic acid (1) was not unexpected. In fact, 1 binds to MMP12 with a Kd of 6.18 mM, its binding mode has been confirmed by X-ray crystallography and the subsequent ITC analysis revealed this fragment as a high enthalpy binder. The first optimization step was realized by a linking strategy connecting the acetyl group of 1 to the terminal amino group of the 4-methoxy-benzenesulfonamide (2) that binds to the S1’ pocket of MMP12 (Figure 2). This modification resulted in a dramatic improvement in the potency that was increased by five orders of magnitude, reaching the Kd of 61 nM. Analyzing the thermodynamic profiles of the linked fragments, the observed improvement in Kd can be traced back to the much improved binding entropy of 3.
Resveratrol inhibits ACHN cells via regulation of histone acetylation
Published in Pharmaceutical Biology, 2020
Lili Dai, Lingyan Chen, Wenjing Wang, Peizheng Lin
Matrix metalloproteinases are considered to be regulatory factors of tumour microenvironment and carcinogenesis (Strbac et al. 2018). The MMPs are involved in the key ECM degradation of proteolytic enzyme family (Curran et al. 2004; Chen KE et al. 2018; Qiao et al. 2018) and are used to disrupt the interaction of cells–cells and cells–ECM by tissue remodelling. Studies have shown that the MMPs, particularly MMP-2 and MMP-9, key members of the MMP family, play a vital role in angiogenesis, invasion and transferring of tumours, including RCC (Ramos et al. 2016; Chen et al. 2017; Liu et al. 2017; Beardo et al. 2019). Therefore, gelatine zymography was performed to determine the activity of MMP-2/-9. We found that resveratrol significantly decreased MMP-2 and MMP-9 expressions. However, one previous research reported that the expression level of MMP-2 in ACHN cells was not affected by resveratrol (Zhao et al. 2018). The difference may be explained by concentrations of resveratrol and action times applied in the two studies. These data indicated that resveratrol could inhibit activity of MMP-2/-9. Studies have shown that MMP-2/-9 activity is associated with histone acetylation (Yeh et al. 2016).