Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis

Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable, 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange between ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivatives was capable of promoting a significant level of ubiquitin-dependent proteolysis. D was capable of forming conjugates with exogenous and endogenous proteins to an extent very similar to that of native ubiquitin, suggesting that its inability to stimulate ubiquitin-dependent proteolysis was due to a defect in a step beyond that of conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP)

Phytohemagglutinin or Concanavalin A-stimulated human T-lymphocytes produce a factor (LYNAP) with potent chemotactic and enzyme degranulating activity in peripheral human neutrophils. Sequence analysis of LYNAP established an apparently novel 72 residue polypeptide structure. Examination of protein data bases showed that LYNAP had about 30% sequence homology with recently characterised connective tissue activating proteins produced by platelets. Furthermore, it was subsequently found that the amino acid sequence is largely the same as that predicted from a cDNA clone derived from mRNA elevated in peripheral human leukocytes stimulated by mitogens.     

Structure elucidation of platelet activating factor derived from human neutrophils

Platelet activating factor (PAF) synthesized by human neutrophils challenged by opsonized zymosan or calcium ionophore was isolated from cells and buffer using Bligh and Dyer extraction following the addition of tracer amounts of tritiated-PAF. The extract was subjected to TLC separation of phospholipid classes, followed by reverse phase HPLC for molecular species separation. All fractions were measured for radioactivity, biological activity and fast atom bombardment mass spectrometry. While the radioactive tracer PAF could be separated into three molecular species, PAF biological activity eluted as a single component which was characterized as 1-O-hexadecyl-2-acetyl-glycero-3-phosphocholine. The lack of molecular species heterogeneity of PAF produced in response to stimuli implies a higher degree of control of biosynthesis than previously suspected.     

Dynamics of the ClpP serine protease: A model for self-compartmentalized proteases

Abstract

ClpP is a highly conserved serine protease present in most bacterial species and in the mitochondria of mammalian cells. It forms a cylindrical tetradecameric complex arranged into two stacked heptamers. The two heptameric rings of ClpP enclose a roughly spherical proteolytic chamber of about 51 Å in diameter with 14 Ser–His–Asp proteolytic active sites. ClpP typically forms complexes with unfoldase chaperones of the AAA+ superfamily. Chaperones dock on one or both ends of the ClpP double ring cylindrical structure. Dynamics in the ClpP structure is critical for its function. Polypeptides targeted for degradation by ClpP are initially recognized by the AAA+ chaperones. Polypeptides are unfolded by the chaperones and then translocated through the ClpP axial pores, present on both ends of the ClpP cylinder, into the ClpP catalytic chamber. The axial pores of ClpP are gated by dynamic axial loops that restrict or allow substrate entry. As a processive protease, ClpP degrades substrates to generate peptides of about 7–8 residues. Based on structural, biochemical and theoretical studies, the exit of these polypeptides from the proteolytic chamber is proposed to be mediated by the dynamics of the ClpP oligomer. The ClpP cylinder has been found to exist in at least three conformations, extended, compact and compressed, that seem to represent different states of ClpP during its proteolytic functional cycle. In this review, we discuss the link between ClpP dynamics and its activity. We propose that such dynamics also exist in other cylindrical proteases such as HslV and the proteasome.     

Kinetics of a model for zymogen activation: the case of high activating enzyme concentrations

Transient phase and steady state equations have been derived for the following enzyme activation mechanism: (formula; see text) in which the concentrations of activating enzyme, E, and substrate, A, are greatly in excess of that of zymogen, Ei. EEi, EEa, EaA and EaY are four intermediates; W is a peptide related from Ei during EEa formation and X and Y are the products of Ea reaction on A. From the general equations, approximate solutions under certain simplifying conditions have been derived. Finally, some formal particular cases of the above mechanism are considered.     

The sulfonimidamide as a novel transition state analog for aspartic acid and metallo proteases.

We have developed a novel strategy for the preparation of tetrahedral transition state analogs for aspartic acid and metallo-proteases based upon the sulfonimidamide functional group. Our best alpha-des-amino dipeptide analog binds at least 100-fold tighter than the corresponding ground state structure (i.e., amide). A previously unpublished five-membered cyclic sulfonimidamide was also synthesized.     

Structure of the ExoS GTPase activating domain

Pseudomonas aeruginosa is an opportunistic bacterial pathogen of great medical relevance. One of its major toxins, exoenzyme S (ExoS), is a dual function protein with a C-terminal Ras-ADP-ribosylation domain and an N-terminal GTPase activating protein (GAP) domain specific for Rho-family proteins. We report here the three-dimensional structure of the N-terminal domain of ExoS determined by X-ray crystallography to 2.4 A resolution. Its fold is all helical with a four helix bundle core capped by additional irregular helices. Loops that are known to interact with Rho-family proteins show very large mobility. Considering the importance of ExoS in Pseudomonas pathogenicity, this structure could be of interest for drug targeting.     

Pseudoconservative transition: a two-state model for the co-operative behavior of oligomeric proteins.

A plausible extension of the two-state concerted model is proposed. This extended two-state model involves at least one state with pairwise asymmetry and consequent heterogeneous binding properties. Such a pseudoconservative transition model accounts in a predictably restricted manner for either positive or negative co-operativity² and for a combination of both in the binding of a single ligand. State and saturation functions are derived. Variation of the Hill number with respect to the extent of binding provides a diagnostic test for the combination of both positive and negative co-operativities in ligand binding. Applications of the model to recent experimental data are discussed.     

Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) participate in virtually all physiological processes. They constitute the largest and most structurally conserved family of signaling molecules. Several class C GPCRs have been shown to exist as dimers in their active form and growing evidence indicates that many, if not all, class A receptors also form dimers and/or higher-order oligomers. High-resolution crystal structures are available only for the detergent-solubilized light receptor rhodopsin (Rho), the archetypal class A GPCR. In addition, Rho is the only GPCR for which the presumed higher-order oligomeric state has been demonstrated, by imaging native disk membranes using atomic force microscopy (AFM). Based on these data and the X-ray structure, an atomic model of Rho dimers has been proposed, a model that is currently scrutinized in various ways. AFM has also been used to measure the forces required to unfold single Rho molecules, thereby revealing which residues are responsible for Rho's stability. Recent functional analyses of fractions from solubilized disk membranes revealed that higher-order Rho oligomers are the most active species. These and other results have enhanced our understanding of GPCR structure and function.     

Zebrafish: a model system for the study of human disease

The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which resemble human clinical disorders. The creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease.     

Purification of a lymphokine: Osteoclast activating factor from human tonsil lymphocytes

Osteoclast activating factor is a lymphokine produced by mitogen-stimulated human lymphocytes. The current studies describe purification to essential homogeneity of the major form of osteoclast activating factor present in supernatants of phytohemagglutinin stimulated lymphocyte cultures. Preliminary chemical and biological characterization of the purified material was carried out. The active factor is a peptide which migrates in polyacrylamide gel electrophoresis as an α-2 fraction in native gels and as a 9,000-dalton species in sodium dodecyl sulfate-urea gels. The purified fraction stimulates bone resorption $\text{in}\text{vitro}$ at doses between 0.1 and 500 ng/ml, with half-maximal stimulation at approximately 1 ng/ml.     

Structure and mechanism of ATP-dependent proteases.

A general paradigm for energy-dependent proteases is emerging: ATP may be used to unfold the substrate and translocate it through a narrow channel within the enzyme into a central proteolytic chamber. Different members of the family present intriguing elaborations on this model.     

An assay for high-sensitivity detection of thrombin activity and determination of proteases activating or inactivating protease-activated receptors

This paper describes the development of galactosidase protease-activated receptor (GPAR) as a recombinant protein obtained by fusion of beta-galactosidase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein allows the detection of thrombin in the sub-picomolar range. A comparative analysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human PAR4 was performed, involving mutated and nonmutated GPAR fusion proteins. Thrombin cleaved GPAR1 (2.6 mol(beta-galactosidase)/(mol(thrombin) * min)), GPAR3 (410 mmol(beta-galactosidase)/(mol(thrombin) * min)), and GPAR4 (4.3 mmol(beta-galactosidase)/(mol(thrombin) * min)) specifically at the proteolytic activation site. A second possible cleavage site for thrombin is present in murine PAR1 and PAR3. Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a marked preference of trypsin for cleavage at the activation site of GPAR2. Chymotrypsin cleaves GPAR1 at a rate (58 mmol(beta-galactosidase)/(mol(thrombin) * min)) that suggests the possibility of chymotryptic inactivation of PAR1. Elastase may inactivate PAR1 and PAR3, but probably not PAR2 and PAR4. Neither activated protein C nor the plasminogen activators cleave any GPAR fusion protein at considerable rates.     

Structure of the tomato bushy stunt virus: a model for protein-RNA interaction

The structure of tomato bushy stunt virus has been investigated by neutron small-angle scattering. Data were collected in aqueous solutions containing various amounts of D₂O, and the radial distribution of both protein and RNA could be computed. The main feature of that distribution is the clustering of protein into two concentric shells separated by about 30 Å, where the density is so low that the polypeptide chain must be extended. Most of the RNA is located between these two protein shells. The implications of that structure for protein-RNA interactions are discussed.     

SUMO-specific proteases: a twist in the tail

The small ubiquitin-like modifier (SUMO) is involved in many cellular processes and is required for normal growth and development in all eukaryotes. Whereas lower eukaryotes have a single version of SUMO, higher eukaryotes have three versions: SUMO-1, -2 and -3. Similarly to most other ubiquitin-like proteins, the primary translation products of the SUMO genes need to be proteolytically processed to expose the C-terminal glycine that will be linked to lysine side chains in substrates. Processing of SUMO precursors is mediated by SUMO-specific proteases that also remove SUMO from modified proteins and depolymerise poly-SUMO chains.     

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.