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Nature Communications Aug 2022We present three classes of chemical zymogens established around the protein cysteinome. In each case, the cysteine thiol group was converted into a mixed disulfide:...
We present three classes of chemical zymogens established around the protein cysteinome. In each case, the cysteine thiol group was converted into a mixed disulfide: with a small molecule, a non-degradable polymer, or with a fast-depolymerizing fuse polymer (Z). The latter was a polydisulfide based on naturally occurring molecule, lipoic acid. Zymogen designs were applied to cysteine proteases and a kinase. In each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only Z could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the active site of the enzyme. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. We also document zymogen exchange reactions whereby the polydisulfide is transferred between the interacting proteins via the "chain transfer" bioconjugation mechanism.
Topics: Cysteine; Disulfides; Enzyme Precursors; Polymers
PubMed: 35982075
DOI: 10.1038/s41467-022-32609-1 -
Current Opinion in Gastroenterology Sep 2011Despite being a subject of much scientific scrutiny, the pathogenesis of acute pancreatitis is still not well understood. This article reviews recent advances in our... (Review)
Review
PURPOSE OF REVIEW
Despite being a subject of much scientific scrutiny, the pathogenesis of acute pancreatitis is still not well understood. This article reviews recent advances in our understanding of acute pancreatitis.
RECENT FINDINGS
Zymogen activation, observed within acini early during acute pancreatitis for a long time, was shown to be sufficient to induce acute pancreatitis. Another key early event, NFκB activation, has previously been shown to induce acute pancreatitis. The relationship between these two key early steps is beginning to be clarified. Mechanisms of zymogen activation - pathologic calcium signaling, pH changes, colocalization and autophagy, and of NFκB activation have been investigated intensively along with potential therapeutic targets both upstream and downstream of these key events. Additional key findings have been elucidation of the role of bioenergetics and the dual role of oxidative stress in acute pancreatitis, recognition of endoplasmic reticulum stress as an early step and the status of duct cells as important entities in pancreatic injury.
SUMMARY
Current findings have provided further insight into the roles and mechanisms of zymogen activation and inflammatory pathways in pancreatic injury. Future studies, which will be of great importance in identifying therapeutic targets, are being undertaken to establish the relative contributions of these pathways during acute pancreatitis.
Topics: Animals; Endoplasmic Reticulum; Energy Metabolism; Enzyme Precursors; Humans; NF-kappa B; Oxidative Stress; Pancreatitis; Signal Transduction
PubMed: 21844752
DOI: 10.1097/MOG.0b013e328349e346 -
Biological Chemistry Oct 2012The astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by... (Review)
Review
The astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by being synthesized as inactive zymogens and also by co-localizing protein inhibitors. The distinct family members consist of N-terminal signal peptides and pro-segments, zinc-dependent catalytic domains, further downstream extracellular domains, transmembrane anchors, and cytosolic domains. The catalytic domains of four astacins and the zymogen of one of these have been structurally characterized and shown to comprise compact ~200-residue zinc-dependent moieties divided into an N-terminal and a C-terminal sub-domain by an active-site cleft. Astacins include an extended zinc-binding motif (HEXXHXXGXXH) which includes three metal ligands and groups them into the metzincin clan of metallopeptidases. In mature, unbound astacins, a conserved tyrosine acts as an additional zinc ligand, which is swung out upon substrate or inhibitor binding in a 'tyrosine switch' motion. Other characteristic structural elements of astacin catalytic domains are three large α-helices and a five-stranded β-sheet, as well as two or three disulfide bonds. The N-terminal pro-segments are variable in length and rather unstructured. They inhibit the catalytic zinc following an 'aspartate-switch' mechanism mediated by an aspartate embedded in a conserved motif (FXGD). Removal of the pro-segment uncovers a deep and extended active-site cleft, which in general shows preference for aspartate residues in the specificity pocket (S1'). Furthermore, astacins undergo major rearrangement upon activation within an 'activation domain,' and show a slight hinge movement when binding substrates or inhibitors. In this review, we discuss the overall architecture of astacin catalytic domains and their involvement in function and zymogenic activation.
Topics: Amino Acid Sequence; Animals; Enzyme Activation; Enzyme Precursors; Evolution, Molecular; Humans; Metalloendopeptidases; Molecular Sequence Data; Protease Inhibitors
PubMed: 23092796
DOI: 10.1515/hsz-2012-0149 -
Blood Aug 1990This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated...
This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl-epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.
Topics: Animals; Binding Sites; Blood Coagulation; Dogs; Enzyme Precursors; Factor X; Fibrinolysis; Immunoassay; Serine Endopeptidases; Tissue Plasminogen Activator
PubMed: 2116928
DOI: No ID Found -
Scientific Reports Apr 2019Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations,...
Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.
Topics: Allosteric Regulation; Chymotrypsin; Crystallography, X-Ray; Enzyme Precursors; Factor Xa; Protein Domains; Protein Stability; Proteolysis; Prothrombin; Structure-Activity Relationship; Thrombin
PubMed: 30992526
DOI: 10.1038/s41598-019-42524-z -
The Biochemical Journal Nov 1998The gastric aspartic proteinases (pepsin A, pepsin B, gastricsin and chymosin) are synthesized in the gastric mucosa as inactive precursors, known as zymogens. The... (Review)
Review
The gastric aspartic proteinases (pepsin A, pepsin B, gastricsin and chymosin) are synthesized in the gastric mucosa as inactive precursors, known as zymogens. The gastric zymogens each contain a prosegment (i.e. additional residues at the N-terminus of the active enzyme) that serves to stabilize the inactive form and prevent entry of the substrate to the active site. Upon ingestion of food, each of the zymogens is released into the gastric lumen and undergoes conversion into active enzyme in the acidic gastric juice. This activation reaction is initiated by the disruption of electrostatic interactions between the prosegment and the active enzyme moiety at acidic pH values. The conversion of the zymogen into its active form is a complex process, involving a series of conformational changes and bond cleavage steps that lead to the unveiling of the active site and ultimately the removal and dissociation of the prosegment from the active centre of the enzyme. During this activation reaction, both the prosegment and the active enzyme undergo changes in conformation, and the proteolytic cleavage of the prosegment can occur in one or more steps by either an intra- or inter-molecular reaction. This variability in the mechanism of proteolysis appears to be attributable in part to the structure of the prosegment. Because of the differences in the activation mechanisms among the four types of gastric zymogens and between species of the same zymogen type, no single model of activation can be proposed. The mechanism of activation of the gastric aspartic proteinases and the contribution of the prosegment to this mechanism are discussed, along with future directions for research.
Topics: Amino Acid Sequence; Animals; Chymosin; Digestion; Enzyme Precursors; Gastric Mucosa; Humans; Models, Molecular; Molecular Sequence Data; Pepsinogen A; Pepsinogen C; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 9794784
DOI: 10.1042/bj3350481 -
Folia Histochemica Et Cytobiologica 2011Cathepsin E belongs to the third class of enzymes - hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites.... (Review)
Review
Cathepsin E belongs to the third class of enzymes - hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites. Cathepsin E is an endopeptidase with substrate specificity similar to that of cathepsin D. In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes. In human cells, loci of the gene of pre-procathepsin E are located on chromosome 1 in the region 1231-32. The catalytic site of cathepsin E is two residues of aspartic acid - Asp96 and Asn281, occurring in amino acid triads with sequences DTG96-98 and DTG281-283. To date, no particular role of cathepsin E in the metabolism of proteins in normal tissues has been found. However, it is known that there are many documented pathological conditions in which overexpression of cathepsin E occurs.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Cathepsin E; Enzyme Precursors; Humans; Molecular Sequence Data; Protein Conformation; Substrate Specificity
PubMed: 22252749
DOI: 10.5603/fhc.2011.0078 -
Biomolecular Concepts Dec 2018Meizothrombin is an active intermediate generated during the proteolytic activation of prothrombin to thrombin in the penultimate step of the coagulation cascade....
Meizothrombin is an active intermediate generated during the proteolytic activation of prothrombin to thrombin in the penultimate step of the coagulation cascade. Structurally, meizothrombin differs from thrombin because it retains the auxiliary Gla domain and two kringles. Functionally, meizothrombin shares with thrombin the ability to cleave procoagulant (fibrinogen), prothrombotic (PAR1) and anticoagulant (protein C) substrates, although its specificity toward fibrinogen and PAR1 is less pronounced. In this study we report information on the structural architecture of meizothrombin resolved by SAXS and single molecule FRET as an elongated arrangement of its individual domains. In addition, we show the properties of a meizothrombin construct analogous to the anticoagulant thrombin mutant W215A/E217A currently in Phase I for the treatment of thrombotic complications and stroke. The findings reveal new structural and functional aspects of meizothrombin that advance our understanding of a key intermediate of the prothrombin activation pathway.
Topics: Amino Acid Substitution; Animals; Anticoagulants; Cell Line; Cricetinae; Cricetulus; Enzyme Precursors; Fibrinogen; Protein C; Protein Domains; Proteolysis; Thrombin
PubMed: 30864392
DOI: 10.1515/bmc-2018-0016 -
PloS One 2018Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in...
Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L.
Topics: Cathepsin H; Cathepsin L; Crystallization; Enzyme Precursors; HEK293 Cells; Humans; Molecular Dynamics Simulation; Protein Conformation; Recombinant Proteins
PubMed: 30044821
DOI: 10.1371/journal.pone.0200374 -
Biochimica Et Biophysica Acta Jan 2010Matrix metalloproteinases (MMPs) are zinc-dependent protein and peptide hydrolases. They have been almost exclusively studied in vertebrates and 23 paralogs are present... (Review)
Review
Matrix metalloproteinases (MMPs) are zinc-dependent protein and peptide hydrolases. They have been almost exclusively studied in vertebrates and 23 paralogs are present in humans. They are widely involved in metabolism regulation through both extensive protein degradation and selective peptide-bond hydrolysis. If MMPs are not subjected to exquisite spatial and temporal control, they become destructive, which can lead to pathologies such as arthritis, inflammation, and cancer. The main therapeutic strategy to combat the dysregulation of MMPs is the design of drugs to target their catalytic domains, for which purpose detailed structural knowledge is essential. The catalytic domains of 13 MMPs have been structurally analyzed so far and they belong to the "metzincin" clan of metalloendopeptidases. These compact, spherical, approximately 165-residue molecules are divided by a shallow substrate-binding crevice into an upper and a lower sub-domain. The molecules have an extended zinc-binding motif, HEXXHXXGXXH, which contains three zinc-binding histidines and a glutamate that acts as a general base/acid during catalysis. In addition, a conserved methionine lying within a "Met-turn" provides a hydrophobic base for the zinc-binding site. Further earmarks of MMPs are three alpha-helices and a five-stranded beta-sheet, as well as at least two calcium sites and a second zinc site with structural functions. Most MMPs are secreted as inactive zymogens with an N-terminal approximately 80-residue pro-domain, which folds into a three-helix globular domain and inhibits the catalytic zinc through a cysteine imbedded in a conserved motif, PRCGXPD. Removal of the pro-domain enables access of a catalytic solvent molecule and substrate molecules to the active-site cleft, which harbors a hydrophobic S(1')-pocket as main determinant of specificity. Together with the catalytic zinc ion, this pocket has been targeted since the onset of drug development against MMPs. However, the inability of first- and second-generation inhibitors to distinguish between different MMPs led to failures in clinical trials. More recent approaches have produced highly specific inhibitors to tackle selected MMPs, thus anticipating the development of more successful drugs in the near future. Further strategies should include the detailed structural characterization of the remaining ten MMPs to assist in achieving higher drug selectivity. In this review, we discuss the general architecture of MMP catalytic domains and its implication in function, zymogenic activation, and drug design.
Topics: Animals; Catalytic Domain; Enzyme Precursors; Humans; Matrix Metalloproteinases; Peptides; Protein Structure, Secondary; Zinc
PubMed: 19374923
DOI: 10.1016/j.bbamcr.2009.04.003