One-step site-directed mutagenesis of the Kex2 protease oxyanion hole

BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.     

Transfer RNA intron processing in the halophilic archaebacteria

An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNA(Trp) precursor. The cleavage reaction produces products having 5' hydroxyl and 2',3' cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNA(Trp) intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.     

Clostridium difficile has two parallel and essential Sec secretion systems

Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.     

The extended cleavage specificity of human thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.     

Kinetic models of translocation, head-on collision, and DNA cleavage by type I restriction endonucleases

Digestion of linear DNA by type I restriction endonucleases is generally activated following the head-on collision of two translocating enzymes. However, the resulting distributions of cleavage loci along the DNA vary with different enzymes; in some cases, cleavage is located in a discrete region midway between a pair of recognition sites while in other cases cleavage is broadly distributed and occurs at nearly every intervening locus. Statistical models for DNA translocation, collision, and cleavage are described that can account for these observations and that are generally applicable to other DNA-based motor proteins. If translocation is processive (stepping forward is significantly more likely than DNA dissociation), then the linear distribution of an ensemble of proteins can be described simply using a Poisson relationship. The pattern of cleavage sites resulting from collision between two processive type I enzymes over a distance d can then be described by a binomial distribution with a standard deviation 0.5 x d1/2. Alternatively, if translocation is nonprocessive (stepping forward or dissociating become equally likely events), the linear distribution is described by a continuum of populated states and is thus extended. Comparisons of model data to the kinetics of DNA translocation and cleavage discount the nonprocessive model. Instead, the observed differences between enzymes are due to asynchronous events that occur upon collision. Therefore, type I restriction enzymes can be described as having processive DNA translocation but, in some cases, nonprocessive DNA cleavage.     

Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.     

Secretion in yeast: preprotein binding to a membrane receptor and ATP- dependent translocation are sequential and separable events in vitro

We have used a cytosol-free assay in which efficient translocation and signal peptide cleavage is achieved when the affinity-purified precursor of OmpA (proOmpA) is diluted out of 8 M urea into a suspension of yeast rough microsomes. This aspect of protein targeting and transport occurs in two discernible steps: (a) in the absence of ATP and cytosolic factors, the precursor binds to the membranes but is not translocated; (b) addition of ATP results in the translocation of the bound precursor and its processing to the mature form. The binding to microsomes of radiolabeled proOmpA is saturable and inhibited by the addition of unlabeled proOmpA but not by mature OmpA or other proteins. The binding of radiolabeled prepro-alpha-factor is also effectively competed by other preproteins, but not by mature ones. Scatchard analysis showed the Kd of proOmpA to be 7.5 X 10(-9) M. Binding is most likely protein mediated as treatment of the microsomes with the protease papain was found to be inhibitory. These results represent the first functional characterization of secretory protein precursor binding to membranes. Alkylation of the microsomes with NEM, washing the membranes with urea or using membranes from the (translocation) mutant ptll at the nonpermissive temperature, did not affect binding, but did eliminate the subsequent ATP-dependent translocation. The ability to subdivide translocation into individual reactions provides a more precise means of determining the membrane components involved in this process.     

Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase

The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its "ancestral" assembly pathway.     

p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.     

ADAMTS1 interacts with, cleaves, and modifies the extracellular location of the matrix inhibitor tissue factor pathway inhibitor-2

ADAMTS1 is an extracellular metalloproteinase known to participate in a variety of biological processes that includes inflammation, angiogenesis, and development of the urogenital system. Many of its functions rely on its catalytic activity, which thus far has been limited to the cleavage of the matrix proteoglycans aggrecan and versican. However, it is likely that other substrates exist. Using a yeast two-hybrid screen, we identified the Kunitz-type inhibitor, tissue factor pathway inhibitor-2 (TFPI-2), as a binding partner of ADAMTS1. The interaction was confirmed by several biochemical and cell-based assays. In addition, our studies revealed alterations in the pattern of TFPI-2-secreted isoforms and in its extracellular location caused by the specific action of ADAMTS1. Interestingly, we found that TFPI-2 is a novel substrate of ADAMTS1. The cleavage removes a protease-sensitive C-terminal region in TFPI-2, altering its binding properties. The proposed role of TFPI-2 as a maintenance factor of extracellular remodeling suggests the indirect function of ADAMTS1 as an additional homeostatic player by its ability to alter the extracellular location of TFPI-2 and, therefore, to disrupt the remodeling machinery, a phenomenon directly associated to pathologies such as atherosclerosis and tumor progression.     

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.     

A test for prorelaxin-processing enzymes using unmodified peptide substrates

Summary Relaxin is a member of the insulin family of proteins. It is produced principally in ovarian cells by processing of its larger precursor, prorelaxin. The enzymes responsible for conversion of prorelaxin to the mature hormone have not yet been elucidated. A rapid and convenient test has been developed to detect prorelaxin-processing enzymes in porcine ovary extracts. Unmodified peptide substrates, which represent the two prorelaxin-processing sites, were chemically synthesised and nanomolar amounts of these substrates were incubated in solution with enzyme preparations. The resultant fragments were resolved using high performance liquid chromatography or capillary electrophoresis and identified by their retention times compared with synthetic standards. This test has been successfully used to identify and characterise a candidate prorelaxin-processing enzyme from chromatographically fractionated porcine ovary extracts. The enzyme was found to be a serine protease with preference for cleavage after tetrabasic sequences and with optimal activity at pH 5.5–6.5.     

Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

Notch and presenilin: a proteolytic mechanism emerges.

Presenilins are needed for proteolytic processing of transmembrane proteins of the Notch/Lin-12 family and for cleavage of the amyloid precursor protein. Accumulating evidence now strongly implicates Presenilin as the catalytic core of a multiprotein complex that executes an unusual intramembranous cleavage of its substrates. In the case of amyloid precursor protein, this cleavage contributes to the generation of small, toxic amyloid peptides that trigger the pathological development of Alzheimer's disease. In the Notch/Lin-12 pathway, Presenilin-mediated cleavage of the receptor is a crucial feature of ligand-induced receptor activation and signal transduction. In this pathway, the Presenilins perform a regulated cleavage event that follows additional processing steps during receptor maturation and ligand-induced ectodomain removal.     

Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase–Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX‐12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase–Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone‐redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.