scholarly journals Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain

2020 ◽  
Vol 21 (22) ◽  
pp. 8687
Author(s):  
Javier Garcia-Pardo ◽  
Sebastian Tanco ◽  
Maria C. Garcia-Guerrero ◽  
Sayani Dasgupta ◽  
Francesc Xavier Avilés ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Domain ◽  
Isotopic Labeling ◽  
Enzymatic Properties ◽  
Peptide Substrates ◽  
Catalytic Domains ◽  
Wnt Proteins ◽  
Strict Requirement ◽  
Small Hydrophobic ◽  
Large Peptide

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1′ position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

scholarly journals Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joanna L. Fox ◽  
Michelle A. Hughes ◽  
Xin Meng ◽  
Nikola A. Sarnowska ◽  
Ian R. Powley ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Analysis ◽  
Cell Fate ◽  
Catalytic Domain ◽  
Caspase 8 ◽  
Type I ◽  
Signaling Complex ◽  
Catalytic Domains ◽  
Regulated Cell Death ◽  
Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

GSK3 takes centre stage more than 20 years after its discovery

2001 ◽  
Vol 359 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Sheelagh FRAME ◽  
Philip COHEN
Keyword(s):  
Substrate Specificity ◽  
Therapeutic Potential ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Wnt Signalling ◽  
Signalling Pathway ◽  
Dimensional Structure ◽  
Entire Body ◽  
Wnt Signalling Pathway ◽  
Wnt Proteins

Identified originally as a regulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now a well-established component of the Wnt signalling pathway, which is essential for setting up the entire body pattern during embryonic development. It may also play important roles in protein synthesis, cell proliferation, cell differentiation, microtubule dynamics and cell motility by phosphorylating initiation factors, components of the cell-division cycle, transcription factors and proteins involved in microtubule function and cell adhesion. Generation of the mouse knockout of GSK3β, as well as studies in neurons, also suggest an important role in apoptosis. The substrate specificity of GSK3 is unusual in that efficient phosphorylation of many of its substrates requires the presence of another phosphorylated residue optimally located four amino acids C-terminal to the site of GSK3 phosphorylation. Recent experiments, including the elucidation of its three-dimensional structure, have enhanced our understanding of the molecular basis for the unique substrate specificity of GSK3. Insulin and growth factors inhibit GSK3 by triggering its phosphorylation, turning the N-terminus into a pseudosubstrate inhibitor that competes for binding with the ‘priming phosphate’ of substrates. In contrast, Wnt proteins inhibit GSK3 in a completely different way, by disrupting a multiprotein complex comprising GSK3 and its substrates in the Wnt signalling pathway, which do not appear to require a ‘priming phosphate’. These latest findings have generated an enormous amount of interest in the development of drugs that inhibit GSK3 and which may have therapeutic potential for the treatment of diabetes, stroke and Alzheimer's disease.

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase

2008 ◽  
Vol 86 (3) ◽  
pp. 251-261 ◽  
Author(s):  
Siying Ye ◽  
Siew Yeen Chai ◽  
Rebecca A. Lew ◽  
David B. Ascher ◽  
Craig J. Morton ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Substrate Affinity ◽  
Angiotensin Iv ◽  
Peptide Substrates ◽  
Fold Reduction ◽  
Leukotriene A4 Hydrolase ◽  
Substrate Cleavage ◽  
Leukotriene A4 ◽  
Key Residues ◽  
Catalytic Cleft

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the ‘entrance’ of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2–9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.

scholarly journals The RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation-promoting factor catalytic domain

2014 ◽  
Vol 70 (8) ◽  
pp. 1022-1026 ◽  
Author(s):  
Francois-Xavier Chauviac ◽  
Giles Robertson ◽  
Doris H. X. Quay ◽  
Claire Bagnéris ◽  
Christian Dumas ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Domain ◽  
Anomalous Scattering ◽  
Molecular Replacement ◽  
Catalytic Domains ◽  
Domain Composition ◽  
Resuscitation Promoting Factors ◽  
Structural Conservation ◽  
High Degree ◽  
Very High

The first structure of the catalytic domain of RpfC (Rv1884), one of the resuscitation-promoting factors (RPFs) fromMycobacterium tuberculosis, is reported. The structure was solved using molecular replacement once the space group had been correctly identified as twinnedP21rather than the apparentC2221by searching for anomalous scattering sites inP1. The structure displays a very high degree of structural conservation with the previously published structures of the catalytic domains of RpfB (Rv1009) and RpfE (Rv2450). This structural conservation highlights the importance of the versatile domain composition of the RPF family.

Synthetic and biological approaches to map substrate specificities of proteases

2019 ◽  
Vol 401 (1) ◽  
pp. 165-182 ◽  
Author(s):  
Shiyu Chen ◽  
Joshua J. Yim ◽  
Matthew Bogyo
Keyword(s):  
Substrate Specificity ◽  
Antigen Processing ◽  
Therapeutic Agents ◽  
Protein Catabolism ◽  
Parasite Invasion ◽  
Peptide Substrates ◽  
Protein Substrates ◽  
Putative Protein ◽  
Natural Protein ◽  
Biological Methods

Abstract Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various disease conditions, such as malignant metastasis, viral infection and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed.

scholarly journals Serpin-derived Peptide Substrates for Investigating the Substrate Specificity of Human Tissue Kallikreins hK1 and hK2

1997 ◽  
Vol 272 (47) ◽  
pp. 29590-29595 ◽  
Author(s):  
Luc Bourgeois ◽  
Michèle Brillard-Bourdet ◽  
David Deperthes ◽  
Maria A. Juliano ◽  
Luiz Juliano ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Tissue ◽  
Peptide Substrates

THROMBIN PROPERTIES

1957 ◽  
Vol 35 (1) ◽  
pp. 743-758 ◽  
Author(s):  
Edward Ronwin
Keyword(s):  
Ionic Strength ◽  
Substrate Specificity ◽  
Enzymatic Activity ◽  
Enzymatic Properties ◽  
Organic Reagents

The enzymatic properties of thrombin have been examined and compared with those of two related enzymes, plasmin and trypsin. The effects of factors such as pH, substrate specificity, ionic strength, cations, anions, and organic reagents on the enzymatic activity of thrombin have been studied. While the three enzymes discussed possess differences, such similarities as were observed are quite striking and permit their classification into one group as tryptic enzymes.

scholarly journals Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium

2001 ◽  
Vol 353 (3) ◽  
pp. 635-644 ◽  
Author(s):  
Hidekazu KUWAYAMA ◽  
Helena SNIPPE ◽  
Mari DERKS ◽  
Jeroen ROELOFS ◽  
Peter J. M. VAN HAASTERT
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalytic Domain ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Kinetic Properties ◽  
Intracellular Camp ◽  
Catalytic Domains ◽  
Dual Specificity

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis–Menten kinetics and a higher affinity for cGMP (Km = 0.22µM) than for cAMP (Km = 145µM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn2+ is preferred to Mg2+, whereas Ca2+ yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC50 of approx. 60µM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.

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