Protease activity in single-chain prekallikrein

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state

ABSTRACTAspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) fromTalaromyces leycettanuswas identified and successfully expressed inPichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions.TlAPA1 shared the highest identity of 70.3 % with the aspartic endopeptidase fromByssochlamys spectabilis(GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. MatureTlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature ofTlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (TlAPA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic proteaseTlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation.IMPORTANCEThe novel aspartic proteaseTlAPA1 was identified fromT. leycettanusand expressed as a zymogen (proTlAPA1) inP. pastoris. Enzymatic characteristics of the mature protein were studied and the specific pattern of zymogen conversion was described. The auto-activation processing of proTlAPA1 proceeded in two stages and an intermediate was identified in this process. These results describe a new subgroup of aspartic protease A1 family and provide insights into a novel mode of activation processing in aspartic proteases.

Modulation of Human Immunodeficiency Virus Type 1 Protease Autoprocessing by Charge Properties of Surface Residue 69

ABSTRACT Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S-transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli. Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PRH69E) folds into active enzymes, and it does so with an apparent Kd (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PRH69E and suggesting a folding defect in the precursor.

Nuclear Localization Sequences in Cytomegalovirus Capsid Assembly Proteins (UL80 Proteins) Are Required for Virus Production: Inactivating NLS1, NLS2, or Both Affects Replication to Strikingly Different Extents

ABSTRACT Scaffolding proteins of spherical prokaryotic and eukaryotic viruses have critical roles in capsid assembly. The primary scaffolding components of cytomegalovirus, called the assembly protein precursor (pAP, pUL80.5) and the maturational protease precursor (pPR, pUL80a), contain two nuclear localization sequences (NLS1 and NLS2), at least one of which is required in coexpression experiments to translocate the major capsid protein (MCP, pUL85) into the nucleus. In the work reported here, we have mutated NLS1 and NLS2, individually or together, in human cytomegalovirus (HCMV, strain AD169) bacmid-derived viruses to test their effects on virus replication. Consistent with results from earlier transfection/coexpression experiments, both single-mutant bacmids gave rise to infectious virus but the double mutant did not. In comparisons with the wild-type virus, both mutants showed slower cell-to-cell spread; decreased yields of infectious virus (3-fold lower for NLS1− and 140-fold lower for NLS2−); reduced efficiency of pAP, pPR, and MCP nuclear translocation (sixfold lower for NLS1− and eightfold lower for NLS2−); increased amounts of a 120-kDa MCP fragment; and reduced numbers of intranuclear capsids. All effects were more severe for the NLS2− mutant than the NLS1− mutant, and a distinguishing feature of cells infected with the NLS2− mutant was the accumulation of large, UL80 protein-containing structures within the nucleus. We conclude that these NLS assist in the nuclear translocation of MCP during HCMV replication and that NLS2, which is unique to the betaherpesvirus UL80 homologs, may have additional involvements during replication.