Spelling suggestions: "subject:"exosite"" "subject:"exosites""
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Identification of potential exosite in cathepsin V necessary for elastin degradationChen, Li Hsuen 11 1900 (has links)
Besides collagen, elastin is the most common connective tissue structural protein in
vertebrates and similar to collagen relatively resistant to non-specific degradation.
Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases,
the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases.
Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with
cathepsin V displaying the highest activity among all known mammalian elastases.
Despite a shared amino acid sequence identity of over 80% between cathepsins V and L
and very similar subsite specificities, only cathepsin V has a potent elastase activity
whereas cathepsin L lacks it. A series of chimera mutants containing various proportions
of cathepsin V and cathepsin L were constructed in an attempt to define a specific region
needed for elastin degradation. It was found that retaining the peptide sequence region
from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity
against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids
112-119) contributes approximately 60% of activity retention. Several additional mutant
proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of
cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of
Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin
L were constructed and evaluated for their elastolytic activities. The results obtained with
those mutant cathepsin proteins support the importance of the amino acid region spanning
the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V,
this peptide region is located below subsite binding pocket S2 and forms a wall-like
barrier which may act as an exosite for the productive binding of cross-linked elastin.
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Identification of potential exosite in cathepsin V necessary for elastin degradationChen, Li Hsuen 11 1900 (has links)
Besides collagen, elastin is the most common connective tissue structural protein in
vertebrates and similar to collagen relatively resistant to non-specific degradation.
Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases,
the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases.
Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with
cathepsin V displaying the highest activity among all known mammalian elastases.
Despite a shared amino acid sequence identity of over 80% between cathepsins V and L
and very similar subsite specificities, only cathepsin V has a potent elastase activity
whereas cathepsin L lacks it. A series of chimera mutants containing various proportions
of cathepsin V and cathepsin L were constructed in an attempt to define a specific region
needed for elastin degradation. It was found that retaining the peptide sequence region
from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity
against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids
112-119) contributes approximately 60% of activity retention. Several additional mutant
proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of
cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of
Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin
L were constructed and evaluated for their elastolytic activities. The results obtained with
those mutant cathepsin proteins support the importance of the amino acid region spanning
the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V,
this peptide region is located below subsite binding pocket S2 and forms a wall-like
barrier which may act as an exosite for the productive binding of cross-linked elastin.
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Identification of potential exosite in cathepsin V necessary for elastin degradationChen, Li Hsuen 11 1900 (has links)
Besides collagen, elastin is the most common connective tissue structural protein in
vertebrates and similar to collagen relatively resistant to non-specific degradation.
Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases,
the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases.
Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with
cathepsin V displaying the highest activity among all known mammalian elastases.
Despite a shared amino acid sequence identity of over 80% between cathepsins V and L
and very similar subsite specificities, only cathepsin V has a potent elastase activity
whereas cathepsin L lacks it. A series of chimera mutants containing various proportions
of cathepsin V and cathepsin L were constructed in an attempt to define a specific region
needed for elastin degradation. It was found that retaining the peptide sequence region
from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity
against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids
112-119) contributes approximately 60% of activity retention. Several additional mutant
proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of
cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of
Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin
L were constructed and evaluated for their elastolytic activities. The results obtained with
those mutant cathepsin proteins support the importance of the amino acid region spanning
the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V,
this peptide region is located below subsite binding pocket S2 and forms a wall-like
barrier which may act as an exosite for the productive binding of cross-linked elastin. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
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Novel Allosteric Inhibitors of ThrombinDesai, Bijoy 24 July 2009 (has links)
Thrombin is a critical enzyme involved in blood coagulation and haemostasis. For this reason the study of its interactions with substrates, inhibitors and modulator is essential. It is also a unique enzyme in the serine protease family because unlike enzymes like trypsin and chymotrypsin its activity is modulated by various endogenous and exogenous ligands. This is due to the presence of “exosites” on the thrombin surface. Exosite II, unlike exosite-I, has not been characterized for its allosteric effect. In order to understand the structural basis of interaction and inhibition of inhibitor 4AS, which possibly interacts with exosite-II, native bovine thrombin crystals and human thrombin crystals grown in presence of 4-AS were prepared. X-ray diffraction data was collected on 4AS soaks of native bovine thrombin as well as human thrombin crystals. The data were phased by molecular replacement using appropriate search models. The structures were refined to R factors of 0.24 and 0.27 for native bovine thrombin-4AS soaks and human thrombin-4AS co-crystals respectively. Examination of a 2Fo-Fc electron density map revealed no density for 4-AS. Low affinity of the inhibitor may be the reason for its absence in the solved structures. In the process of solving these structures, unliganded native bovine thrombin in a new crystal form, previously unreported in literature, was solved. The structure shows an overall topology similar to that found in previously published thrombin molecules. Examination of the crystal packing shows that the exosite-II is solvent exposed. This crystal form can be used in the future to study interaction of exosite-II ligands. To characterize the interaction of sucrose octasulfate with thrombin, which may interact with thrombin exosite-II, fluorimetric equilibrium binding titrations were performed using the active site fluorescent probe para-amino benzamidine. At physiological salt concentrations, the KD was found to be ~22 μM, which is lower than heparin fragment of corresponding length. The higher affinity was attributed to the high charge density of the ligand. Measurement of KD at different salt concentrations showed a significant amount of contribution to the binding energy from ionic interactions. Based on the salt dependence experiments, the number of charged interactions per sucrose octasulfate molecule interacting with thrombin was found to be 3.5. Competitive experiments of sucrose octasulfate with FDs (a sulfated dehydro-polymer being investigated in the lab for its anticoagulant properties) for inhibition of thrombin activity showed competitive effects that did not appear to follow Dixon-Webb competitive phenomenon. It was found that sucrose octasulfate itself is a weak inhibitor of thrombin. To investigate the mode of interaction, co-crystals of sucrose-ocasulfate complexed with thrombin were prepared. High resolution data (2.2 Å) was collected. The structure solved using this data showed weak density for two sucrose octasulfate molecules. Sucrose octasulfate was modeled into this density and refined. The refined structure shows that two sucrose octasulfate molecules bind to two thrombin monomers of the asymmetric unit at exosite-II. One of the sucrose octasulfate molecules interacts with both monomers, and could be present as an artifact of crystal packing. The second molecule interacts with exosite-II of only one of the thrombin monomers. The key residues involved in the interaction are Lys236, His91, Arg93 and Arg101. The thrombin-sucrose octasulfate structure does not show any major deviation from unliganded structure. It is possible that the conformational change may have been masked due to crystal packing. Characterization of this novel interaction mode of sucrose octasulfate interaction with thrombin adds one more candidate to the list of compounds that interact with exosite-II in a manner very similar to heparin, but unlike heparin can inhibit thrombin activity.
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Molecular mechanisms of vaspin action: from adipose tissue to skin and bone, from blood vessels to the brainWeiner, Juliane, Zieger, Konstanze, Pippel, Jan, Heiker, John T. 27 January 2020 (has links)
Visceral adipose tissue derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that
were specifically expressed or overexpressed in the intra abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and ‐glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes. The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.
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Kallikrein-related peptidase 14 is the second KLK protease targeted by the serpin vaspinUlbricht, David, Tindall, Catherine A., Oertwig, Kathrin, Hanke, Stefanie, Sträter, Norbert, Heiker, John T. 27 January 2020 (has links)
Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multispecific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.
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Probing Allosteric, Partial Inhibition of Thrombin Using Novel AnticoagulantsVerespy, Stephen S, III 01 January 2016 (has links)
Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition.
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