Peptidases and proteases of Escherichia coli and Salmonella typhimurium

A number of peptidases and proteases have been identified in Escherichia coli. Although their specific physiological roles are often not known, some of them have been shown to be involved in: the maturation of nascent polypeptide chains; the maturation of protein precursors; the signal peptide processing of exported proteins; the degradation of abnormal proteins; the use of small peptides as nutrients; the degradation of colicins; viral morphogenesis; the inactivation of some regulatory proteins for which a limited lifetime is a physiological necessity. Some of these enzymes act in concert to carry out specific functions. At present, twelve peptidases and seventeen proteases have been characterized. The specificity for only a few of them is known. The possible roles and the properties of these enzymes are discussed in this review.     

Dissection of key determinants of cleavage activity in signal peptidase III (SPaseIII) PibD

In Archaea, type IV prepilins and prearchaellins are processed by designated signal peptidase III (SPaseIII) prior to their incorporation into pili and the archaellum, respectively. These peptidases belong to the family of integral membrane aspartic acid proteases that contain two essential aspartate residues of which the second aspartate is located in a conserved GxGD motif. To this group also bacterial type IV prepilin peptidases, Alzheimer disease-related secretases, signal peptide peptidases and signal peptide peptidase-like proteases in humans belong. Here we have performed detailed in vivo analyses to understand the cleavage activity of PibD, SPaseIII from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using an already established in vivo heterologous system cleavage assay, we could successfully identify the key amino acid residues essential for catalysis of PibD. Furthermore, in trans complementation of a pibD S. acidocaldarius deletion mutant with PibD variants having substituted key amino acids has consolidated our observations of the importance of these residues in catalysis. Based on our data, we propose to re-define class III peptidases/type IV prepilin/prearchaellin peptidases as GxHyD group (rather than GxGD) of proteases [Hy-hydrophobic amino acid].     

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Structural principles of intramembrane proteases

Intramembrane proteases are present in most organisms, and are used by cells to send signal across membranes, to activate growth factors, and to accomplish many other tasks that are beyond the capability of their soluble cousins. These enzymes specialize in cleaving peptide bonds that are normally embedded in cell membranes. They contain multiple membrane-spanning segments, and their catalytic residues are often found within these hydrophobic domains. In the past year, a number of important papers have been published that began to address the structural features of these membrane proteins by X-ray crystallography, electron microscopy, and biochemical methods, including the first report of an intramembrane protease crystal structure, that of Escherichia coli GlpG. Taken together, these studies started to reveal patterns of how intramembrane proteases are constructed, how waters are supplied to the membrane-embedded active site, and how membrane protein substrates interact with them.     

Type I signal peptidases of Gram-positive bacteria

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.     

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro-sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.     

Can the topological distribution of membrane spanning amino acid residues be responsible for the recognition of signal peptides by signal peptide peptidases?

Signal peptides are selectively recognized and degraded by membrane associated proteases called as signal peptide peptidases. The hydrolysis of the signal peptide occurs only after its cleavage from the precursor. The possible reasons for this selectivity have been investigated. The results indicate that in signal peptides, leucine residues are clustered to a large extent on the same side of the membrane spanning alpha helix as the polar residues, but are distinctly separated along the length of the axis. Such topological differences in the distribution of amino acids on the surface of the membrane spanning alpha helix may play a crucial role in selective degradation of signal peptides.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases

Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water-requiring hydrolysis reaction. These enzymes include site-2 protease, gamma-secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent-solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins.     

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D₂O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.     

Structural Biology of Presenilins and Signal Peptide Peptidases

Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of γ-secretase, a protease responsible for the generation of amyloid-β peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.     

Signal peptide peptidases: A family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins

Five genes encode the five human signal peptide peptidases (SPPs), which are intramembrane-cleaving aspartyl proteases (aspartyl I-CLiPs). SPPs have been conserved through evolution with family members found in higher eukaryotes, fungi, protozoa, arachea, and plants. SPPs are related to the presenilin family of aspartyl I-CLiPs but differ in several key aspects. Presenilins (PSENs) and SPPs both cleave the transmembrane region of membrane proteins; however, PSENs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. Though the overall homology between SPPs and PSENs is minimal, they are multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their COOH-termini. They differ in that the active site YD and GxGD containing transmembrane domains of SPPs are inverted relative to PSENs, thus, orienting the active site in a consistent topology relative to the substrate. At least two of the human SPPs (SPP and SPPL3) appear to function without additional cofactors, but PSENs function as a protease, called γ-secretase, only when complexed with Nicastrin, APH-1 and Pen-2. The biological roles of SPP are largely unknown, and only a few endogenous substrates for SPPs have been identified. Nevertheless there is emerging evidence that SPP family members are highly druggable and may regulate both essential physiologic and pathophysiologic processes. Further study of the SPP family is needed in order to understand their biological roles and their potential as therapeutic targets.     

超嗜热古菌的ATP非依赖型蛋白酶和肽酶研究进展

蛋白酶和肽酶在超嗜热古菌的营养代谢、蛋白质转换与加工以及蛋白质质量控制等重要生物学过程中发挥关键作用。超嗜热古菌蛋白酶和肽酶具有优良的热稳定性和高温活性,是研究蛋白质耐热分子机制和酶行使功能上限温度等科学问题的理想材料,同时也具有重要的工业应用价值。本文对超嗜热古菌的ATP非依赖型蛋白酶和肽酶的种类、功能、催化特性、热稳定机制以及应用前景进行综述与分析。     

Cryptides: functional cryptic peptides hidden in protein structures

Peptidergic hormones, neurotransmitters, and neuromodulators are extracellular signaling molecules that play central roles in physiological signal transmissions between various cells, tissues, and organs. These factors are primarily translated as inactive precursor proteins according to the genetic information. These precursor proteins are then cleaved by various proteases including signal peptidases and processing enzymes to produce matured bioactive factors. During these processes, various fragmented peptides are also produced from the same precursor proteins. Such fragmented peptides may have various unexpected biological activities that have not been identified yet because these peptides are considered to be produced and released along with mature factors at the same secretary pathways. Recently, we found that various fragmented peptides of mitochondrial proteins that are produced during the maturation processes, such as fragments of cytochrome c oxidase, activate neutrophils whose functions are distinct from their parent proteins. These findings suggest the existence of many different functional peptides whose functions have not been identified yet. These unidentified peptides may play a variety of roles in various regulatory mechanisms, and therefore, they are expected to provide novel regulatory and signaling mechanisms, "Peptide World".     

Falcipain cysteine proteases of malaria parasites: An update

BackgroundThe malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review.Scope of reviewThis review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites.Major conclusionsBased on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites.General Significance. Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.