N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

2004 ◽  
Vol 32 (5) ◽  
pp. 764-765 ◽  
Author(s):  
O. Plíhal ◽  
J. Sklenář ◽  
J. Kmoníčková ◽  
P. Man ◽  
P. Pompach ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Enzymic Activity ◽  
Enzyme Activation ◽  
Protein Architecture ◽  
Fungal Enzyme ◽  
Hexa Gene ◽  
Catalytic Unit ◽  
Complex Protein ◽  
The Stability ◽  
Inhibition Studies

β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.

In Silico and in Vitro Evaluation of Deamidation Effects on the Stability of the Fusion Toxin DAB389IL-2

2019 ◽  
Vol 16 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Nasrin Zarkar ◽  
Mohammad Ali Nasiri Khalili ◽  
Fathollah Ahmadpour ◽  
Sirus Khodadadi ◽  
Mehdi Zeinoddini
Keyword(s):  
In Silico ◽  
Catalytic Domain ◽  
Md Simulations ◽  
Special Importance ◽  
Native Form ◽  
Vitro Experiment ◽  
In Vitro Experiment ◽  
Pharmaceutical Protein ◽  
The Stability

Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

scholarly journals Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7)

2016 ◽  
Vol 165 ◽  
pp. 7-17 ◽  
Author(s):  
Fan Meng ◽  
Hao Yang ◽  
Colin Jack ◽  
Huaqun Zhang ◽  
Abraham Moller ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Zinc Binding ◽  
Inhibition Studies

scholarly journals The kinetoplastid-specific phosphatase KPP1 attenuates PLK activity to facilitate flagellum inheritance in Trypanosoma brucei

2021 ◽  
Vol 14 (669) ◽  
pp. eabc6435
Author(s):  
Tai An ◽  
Huiqing Hu ◽  
Ziyin Li
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Complex Structure ◽  
Cell Communication ◽  
Specific Protein ◽  
Activation Loop ◽  
Human Parasite ◽  
Complex Protein

Trypanosoma brucei, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr125 by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr198 in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr125 in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.

scholarly journals Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity

2004 ◽  
Vol 378 (3) ◽  
pp. 727-734 ◽  
Author(s):  
Paul C. EVANS ◽  
Huib OVAA ◽  
Maureen HAMON ◽  
Peter J. KILSHAW ◽  
Svetlana HAMM ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Catalytic Domain ◽  
Cysteine Proteases ◽  
Cellular Proteins ◽  
Tumour Necrosis Factor Receptor ◽  
Specific Substrate ◽  
Polyubiquitin Chains ◽  
Finger Protein ◽  
The Stability

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which ‘trim’ polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-κB (nuclear factor κB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IκBα (inhibitor of κB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKγ (IκB kinase γ)/NEMO (NF-κB essential modifier) signal adaptors relies on their modification with ‘nonclassical’ forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-κB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.

scholarly journals A protein architecture guided screen for modification dependent restriction endonucleases

2019 ◽  
Vol 47 (18) ◽  
pp. 9761-9776 ◽  
Author(s):  
Thomas Lutz ◽  
Kiersten Flodman ◽  
Alyssa Copelas ◽  
Honorata Czapinska ◽  
Megumu Mabuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Dna Methyltransferase ◽  
Catalytic Domain ◽  
Dna Methyltransferases ◽  
Restriction Endonucleases ◽  
Endonuclease Activity ◽  
Protein Architecture ◽  
Escherichia Coli Cells ◽  
Dependent Activity

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

scholarly journals PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization

2003 ◽  
Vol 370 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Miles D. HOUSLAY ◽  
David R. ADAMS
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Specific Interaction ◽  
Cellular Functions ◽  
Intracellular Targeting ◽  
Anchoring Proteins ◽  
Extracellular Signal ◽  
Catalytic Unit ◽  
Dependent Protein ◽  
Regulatory Functions

cAMP is a second messenger that controls many key cellular functions. The only way to inactivate cAMP is to degrade it through the action of cAMP phosphodiesterases (PDEs). PDEs are thus poised to play a key regulatory role. PDE4 cAMP-specific phosphodiesterases appear to have specific functions with selective inhibitors serving as potent anti-inflammatory agents. The recent elucidation of the structure of the PDE4 catalytic unit allows for molecular insight into the mode of catalysis as well as substrate and inhibitor selectivity. The four PDE4 genes encode over 16 isoforms, each of which is characterized by a unique N-terminal region. PDE4 isoforms play a pivotal role in controlling functionally and spatially distinct pools of cAMP by virtue of their unique intracellular targeting. Targeting occurs by association with proteins, such as arrestins, SRC family tyrosyl kinases, A-kinase anchoring proteins ('AKAPs') and receptor for activated C kinase 1 ('RACK1'), and, in the case of isoform PDE4A1, by a specific interaction (TAPAS-1) with phosphatidic acid. PDE4 isoforms are ‘designed’ to be regulated by extracellular-signal-related protein kinase (ERK), which binds to anchor sites on the PDE4 catalytic domain that it phosphorylates. The upstream conserved region 1 (UCR1) and 2 (UCR2) modules that abut the PDE4 catalytic unit confer regulatory functions by orchestrating the functional outcome of phosphorylation by cAMP-dependent protein kinase ('PKA') and ERK. PDE4 enzymes stand at a crossroads that allows them to integrate various signalling pathways with that of cAMP in spatially distinct compartments.

scholarly journals Yellow fever virus NS3 protease: peptide-inhibition studies

2007 ◽  
Vol 88 (8) ◽  
pp. 2223-2227 ◽  
Author(s):  
Kristina Löhr ◽  
John E. Knox ◽  
Wai Yee Phong ◽  
Ngai Ling Ma ◽  
Zheng Yin ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Homology Model ◽  
Enzymic Activity ◽  
Fever Virus ◽  
Catalytically Active ◽  
Relationship Of ◽  
Ns3 Protease ◽  
Inhibition Studies ◽  
Factor Sequence

A recombinant form of yellow fever virus (YFV) NS3 protease, linked via a nonapeptide to the minimal NS2B co-factor sequence (CF40-gly-NS3pro190), was expressed in Escherichia coli and shown to be catalytically active. It efficiently cleaved the fluorogenic tetrapeptide substrate Bz-norleucine-lysine-arginine-arginine-AMC, which was previously optimized for dengue virus NS2B/3 protease. A series of small peptidic inhibitors based on this substrate sequence readily inhibited its enzymic activity. To understand the structure–activity relationship of the inhibitors, they were docked into a homology model of the YFV NS2B/NS3 protease structure. The results revealed that the P1 and P2 positions are most important for inhibitor binding, whilst the P3 and P4 positions have much less effect. These findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.

scholarly journals A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

2020 ◽  
Vol 168 (5) ◽  
pp. 515-533
Author(s):  
Jose M González de Cózar ◽  
Maria Carretero-Junquera ◽  
Grzegorz L Ciesielski ◽  
Sini M Miettinen ◽  
Markku Varjosalo ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Shotgun Proteomics ◽  
Binding Domain ◽  
The Novel ◽  
Binding Model ◽  
Interaction Partners ◽  
The Stability ◽  
Deletion Series

Abstract In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

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