C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.     

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.     

Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10

Neprilysin (NEP) consists of 749 amino acids with two conserved cysteines (734, 746) and a putative CAAX motif (residues 746-749, CRVW) at the C-terminus. To investigate the role of the C-terminal conserved cysteine residues, three NEP mutants (C734S, C746S, and double mutant C734S/C746S) were constructed by use of site-directed mutagenesis. Western blot analysis of lysates of transfected cells revealed the presence of three NEP forms in wild type and mutants with a different glycosylation pattern. Point mutations of C734 as well as C746 by serine dramatically diminished the plasma membrane association of NEP as detected by flow cytometry and laser scanning microscopy. Endoprotease enzyme activity was slightly diminished in the C746S-NEP variant and was not detectable in the C734S-form of NEP suggesting a pivotal role of the C734 in the proper folding of the enzyme. Prenylation of NEP was not detected in an in vivo assay.     

Regulation of bacterial protease activity

Proteases, also referred to as peptidases, are the enzymes that catalyse the hydrolysis of peptide bonds in polipeptides. A variety of biological functions and processes depend on their activity. Regardless of the organism’s complexity, peptidases are essential at every stage of life of every individual cell, since all protein molecules produced must be proteolytically processed and eventually recycled. Protease inhibitors play a crucial role in the required strict and multilevel control of the activity of proteases involved in processes conditioning both the physiological and pathophysiological functioning of an organism, as well as in host-pathogen interactions. This review describes the regulation of activity of bacterial proteases produced by dangerous human pathogens, focusing on the Staphylococcus genus.     

The role of protease activity in ErbB biology

Proteases are now recognized as having an active role in a variety of processes aside from their recognized metabolic role in protein degradation. Within the ErbB system of ligands and receptors, proteases are known to be necessary for the generation of soluble ligands from transmembrane precursors and for the processing of the ErbB4 receptor, such that its intracellular domain is translocated to the nucleus. There are two protease activities involved in the events: proteases that cleave within the ectodomain of ligand (or receptor) and proteases that cleave the substrate within the transmembrane domain. The former are the ADAM proteases and the latter are the γ-secretase complex and the rhomboid proteases. This review discusses the roles of each of these protease systems within the ErbB system.     

Requirement of N- and C-terminal regions for enzymatic activity of human T-cell leukemia virus type I protease.

The requirement of N- and C-terminal regions for the enzymatic activity of human T-cell leukemia virus type I (HTLV-I) protease was investigated using a series of deletion mutants. The activity was analyzed by autoprocessing of the protease itself or by processing of the gag p53 precursor. The deletional analyses indicated that Asp38-Gly152 with an additional Met-Pro sequence at the N-terminus was probably sufficient for the enzymatic activity, although the mature HTLV-I protease consists of Pro33-Leu157. A molecular model of HTLV-I protease, which was constructed by comparison with the structure of Rous sarcoma virus protease, predicted that Pro33-Leu37 and Gly143-Leu147 would form a beta-sheet. Our experimental results and the model structure suggest that (a) five amino acids in the N-terminal region (Pro33-Leu37), which are thought to be involved in the beta-sheet, are not crucial for the enzymatic activity; (b) Pro153-Leu157 is not necessary but Pro148-Gly152 is important for the enzymatic activity, in addition to Gly143-Leu147 involved in the beta-sheet.     

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening.     

Backbone (15)N relaxation analysis of the N-terminal domain of the HTLV-I capsid protein and comparison with the capsid protein of HIV-1

Human T-cell leukemia virus type 1 (HTLV-I) is an oncogenic retrovirus that exhibits specific tropism for human T-cells. The capsid (CA) proteins of retroviruses share highly conserved secondary and tertiary structures. However, they can form quaternary structures (assembled cores) that are conical (e.g., the lentivirus subgroup, including HIV) or spherical (e.g., the oncovirus subgroup, including HTLV). The intrinsic features that drive these differences are not understood. So far, only structural studies have been used as a basis for comparison. Dynamics may play a role in particle formation. High-resolution nuclear magnetic resonance (NMR) (15)N relaxation data (T(1), T(1rho), and NOE) have been used to characterize the backbone dynamics of the N-terminal domain (NTD) of the oncovirus HTLV-I and to compare with the CA NTD of HIV-1. Large variations in the (15)N heteronuclear NOEs and transversal relaxation rates for individual residues are consistent with the bundle RMSD of the previously calculated NMR structures. The beta-hairpin and CyP-A loop exhibit different mobility in HTLV-I and HIV-1. The overall hydrodynamic property of the HTLV-I capsid NTD is quite distinct from the HIV-1.     

Thermodynamic and functional characterization of protein W from bacteriophage lambda. The three C-terminal residues are critical for activity

Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions. Composed of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA. To study the structural and functional properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus. The purified wild type fusion proteins were found to be stably folded and biologically active. The protein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism. Circular dichroism studies indicate that the protein is 47% helical, with a T(m) of 71.3 degrees C, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C. Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also designed destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli.     

The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.

Cytochrome b5 is an integral membrane protein located on the outer surface of the endoplasmic reticulum (ER). This cytochrome is considered to be synthesized on free ribosomes and to be inserted post-translationally into the ER membrane, without participation of a signal recognition particle. To elucidate the signal responsible for targeting of cytochrome b5 to the ER membrane in vivo, DNAs encoding various derivatives of the cytochrome were constructed and introduced into cultured mammalian COS cells, and the subcellular distributions of the derivatives expressed in the cells were then analyzed. The deletion of more than 11 amino acid residues at the carboxy-terminal end of cytochrome b5 abolished the targeting and anchoring of the cytochrome to the ER membrane. Fusion proteins consisting of the carboxy-terminal 10 amino acid residues of cytochrome b5 and passenger proteins with the hydrophobic portion could be localized in the ER membrane. Thus, the last 10 amino acid residues of cytochrome b5 carry information necessary for the cytochrome to be targeted to the ER membrane.     

The flavivirus protease as a target for drug discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.     

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

The peptide N-acetyl-CRATKML-amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61-64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl-containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin-converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2-mercapto-3-phenylpropionyl yielded a peptide with K(i) of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1' arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.     

Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.     

Catalytic activity of Teflon particle-immobilized protease in aqueous solution

-Chymotrypsin (Chy) was entrapped in polytetrafluoroethylene (PTFE) particles. The entrapped enzyme showed twofold catalytic activity for amino acid ester hydrolysis in aqueous solution than free enzyme. The Chy/PTFE particles also catalyzed the peptide synthesis in aqueous solution with a yield of 14%. Both the synthetic and the hydrolytic activities of the entrapped enzyme were enhanced as compared with the free enzyme. The PTFE matrix should provide the enzyme molecules by creating a hydrophobic environment which results in enhanced peptide synthesis in aqueous solution.     

Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification

Cephalosporin acylase (CA) is a recently identified N-terminal hydrolase. It is also a commercially important enzyme in producing 7-aminocephalosporanic acid (7-ACA), a backbone chemical in synthesizing semi-synthetic cephalosporin antibiotics. CA is translated as an inactive single chain precursor, being post-translationally modified into an active enzyme. The post-translational modification takes place in two steps. The first intramolecular autocatalytic proteolysis takes place at one end of the spacer peptide by a nucleophilic Ser or Thr, which in turn becomes a new N-terminal Ser or Thr. The second intermolecular modification cleaves off the other end of the spacer peptide by another CA. Two binary structures in complex with glutaryl-7-ACA (the most favored substrate of CAs) and glutarate (side chain of glutaryl-7-ACA) were determined, and they revealed the detailed interactions of glutaryl-7-ACA with the active site residues (Y. Kim and W. G. J. Hol (2001) Chem. Biol., in press). In this report: 1) we have mutated key active site residues into nonfunctional amino acids, and their roles in catalysis were further analyzed; 2) we performed mutagenesis studies indicating that secondary intermolecular modification is carried out in the same active site where deacylation reaction of CA occurs; and 3) the cleavage site of secondary intermolecular modification by another CA was identified in the spacer peptide using mutational analysis. Finally, a schematic model for intermolecular cleavage of CA is proposed.     

Aromatic rings of tyrosine residues at adenine nucleotide binding sites of the beta subunits of F1-ATPase are not necessary for ATPase activity

Using site-directed mutagenesis, Tyr-307, Tyr-341, or Tyr-364, supposedly located at the adenine nucleotide binding site(s) of the beta subunits of F1-ATPase from the thermophilic bacterium PS3, was replaced with Phe or Cys. The alpha 3 beta 3 complexes reconstituted from the alpha subunits and individual mutant beta subunits hydrolyzed ATP. Thus, neither the hydroxyl groups nor the aromatic rings in these positions are required for ATPase activity of F1-ATPase.     

Urease-gelatin interdigitated microelectrodes for the conductometric determination of protease activity

Conductometric microbiosensors for the determination of trypsin were elaborated via the modification of microfabricated interdigitated gold electrodes by a cross-linked urease/BSA coating covered by a gelatin film. The resulting microelectrodes were exposed to different trypsin concentrations ranging from 100pg/mL to 1mg/mL (1mU/mL to 10,000U/mL) for selective proteolytic degradation of the gelatin film. Then, the conductometric response of the microbiosensors to urea (33muM) was recorded as a function of the trypsin concentration, the gelatin amount (8-80ng) and the incubation time (40s, 100min). The optimum incubation time for each trypsin concentration was determined leading to a detection limit of 100pg/mL (1mU/mL). In these optimized conditions, the proof of concept of this sensitive, disposable, low-cost and label-free trypsin biosensors based on a conductometric transducer was demonstrated for the first time.     

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.     

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.