scholarly journals Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

2021 ◽  
Author(s):  
Jie Yang ◽  
Albert A. Song ◽  
R. Luke Wiseman ◽  
Gabriel C. Lander
Keyword(s):  
Serine Protease ◽  
Alpha Helix ◽  
Lon Protease ◽  
Structural Elements ◽  
Active Conformation ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Enzymatic Function ◽  
Biochemical Studies ◽  
Lon Proteases

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a $\sim$50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a $\sim$15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1 enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

scholarly journals Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nis V. Nielsen ◽  
Elfie Roedel ◽  
Dipankar Manna ◽  
Michael Etscheid ◽  
Jens Preben Morth ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Serine Protease ◽  
Active Site ◽  
Factor Vii ◽  
Protease Domain ◽  
Functional Changes ◽  
E Coli ◽  
Catalytic Activation ◽  
Serine Protease Domain ◽  
Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

scholarly journals The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells

2005 ◽  
Vol 388 (3) ◽  
pp. 967-972 ◽  
Author(s):  
Susan WILSON ◽  
Brett GREER ◽  
John HOOPER ◽  
Andries ZIJLSTRA ◽  
Brian WALKER ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Serine Protease ◽  
G Protein ◽  
Calcium Mobilization ◽  
Prostate Cancer Cells ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Pre Treatment ◽  
G Protein Coupled

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1–4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.

scholarly journals A novel cleavage site within the potato leafroll virus P1 polyprotein

2007 ◽  
Vol 88 (5) ◽  
pp. 1620-1623 ◽  
Author(s):  
Xuejun Li ◽  
Claire Halpin ◽  
Martin D. Ryan
Keyword(s):  
Serine Protease ◽  
Cleavage Site ◽  
Mutational Analysis ◽  
Peptide Sequencing ◽  
Potato Leafroll Virus ◽  
Proteolytic Processing ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Sequencing Studies ◽  
Leafroll Virus

To study the proteolytic processing of the potato leafroll virus replicase proteins, the multidomain P1 protein with a c-myc epitope tag attached at the N terminus was expressed in insect cells by using the baculovirus system. Western blotting showed that P1 was cleaved at a site upstream of the serine protease domain, in addition to the cleavage site downstream of the protease domain. Mutational analysis showed that the serine protease domain within P1 was responsible for this cleavage. To characterize this novel cleavage site further, a portion of the P1 protein comprising the protease domain and the two cleavage sites was expressed in Escherichia coli. A similar cleavage event was observed in bacteria and was abolished when the P1 protease was inactivated by mutation. Peptide-sequencing studies indicated that this cleavage occurred at a Glu/Arg junction, separating the N-terminal 204 residues from the serine protease domain of P1.

scholarly journals Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity

2005 ◽  
Vol 390 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Roman Szabo ◽  
Sarah Netzel-Arnett ◽  
John P. Hobson ◽  
Toni M. Antalis ◽  
Thomas H. Bugge
Keyword(s):  
Cell Surface ◽  
Serine Protease ◽  
Transmembrane Domain ◽  
Density Lipoprotein ◽  
Biologically Active ◽  
Chromosome 1 ◽  
Mrna Levels ◽  
Protease Domain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Serine Protease Domain

We report in the present study the bioinformatic identification, molecular cloning and biological characterization of matriptase-3, a novel membrane-anchored serine protease that is phylogenetically preserved in fish, birds, rodents, canines and primates. The gene encoding matriptase-3 is located on syntenic regions of human chromosome 3q13.2, mouse chromosome 16B5, rat chromosome 11q21 and chicken chromosome 1. Bioinformatic analysis combined with cDNA cloning predicts a functional TTSP (type II transmembrane serine protease) with 31% amino acid identity with both matriptase/MT-SP1 and matriptase-2. This novel protease is composed of a short N-terminal cytoplasmic region followed by a transmembrane domain, a stem region with one SEA, two CUB and three LDLRa (low-density lipoprotein receptor domain class A) domains and a C-terminal catalytic serine protease domain. Transcript analysis revealed restricted, species-conserved expression of matriptase-3, with the highest mRNA levels in brain, skin, reproductive and oropharyngeal tissues. The full-length matriptase-3 cDNA directed the expression of a 90 kDa N-glycosylated protein that localized to the cell surface, as assessed by cell-surface biotin labelling. The purified activated matriptase-3 serine protease domain expressed in insect cells hydrolysed synthetic peptide substrates, with a strong preference for Arg at position P1, and showed proteolytic activity towards several macromolecular substrates, including gelatin, casein and albumin. Interestingly, activated matriptase-3 formed stable inhibitor complexes with an array of serpins, including plasminogen activator inhibitor-1, protein C inhibitor, α1-proteinase inhibitor, α2-antiplasmin and antithrombin III. Our study identifies matriptase-3 as a novel biologically active TTSP of the matriptase subfamily having a unique expression pattern and post-translational regulation.

scholarly journals Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice

2021 ◽  
Author(s):  
Ferruh Artunc ◽  
Bernhard N. Bohnert ◽  
Jonas C. Schneider ◽  
Tobias Staudner ◽  
Florian Sure ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Sodium Channel ◽  
Serine Protease ◽  
Cleavage Site ◽  
Factor Vii ◽  
Sodium Retention ◽  
Protease Domain ◽  
Proteolytic Activation ◽  
Serine Protease Domain ◽  
Epithelial Sodium Channel Enac

AbstractProteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2−/−) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2−/− mice. However, Habp2−/− mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2−/− mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

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