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Reports on the 2003 Paper of the Year Award given to Dieter Hoffman for his study "Rapid Enzymatic Test for Phenotypic HIV Protease Drug Resistance," published in 2003 issue of the journal "Biological Chemistry." Academic background of Hoffman; Overview of the study; Significance of the study to biochemistry.

Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various disease conditions, such as malignant metastasis, viral infection and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed. [ABSTRACT FROM AUTHOR]

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers. [ABSTRACT FROM AUTHOR]

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers. [ABSTRACT FROM AUTHOR]

β-site APP-cleaving enzyme (BACE1) cleaves the wild type (WT) β-site very slowly ( kcat /Km: 46.6 m-1 s-1). Therefore we searched for additional β-secretases and identified three cathepsins that split the WT β-site much faster. Human cathepsin S cleaves the WT β-site ( kcat /Km: 54 700 m-1 s-1) 1170-fold faster than BACE1 and cathepsins B and L are 440- and 74-fold faster than BACE1, respectively. These cathepsins split two bonds flanking the WT β-site (K-MD-A), where the K-M bond (85%) is cleaved more efficiently than the D-A bond (15%). Cleavage at the major K-M bond yields Aβ (amyloid β-peptide) extended by N-terminal Met that should be removed to generate Aβ initiated by Asp1. The activity of cytosol and microsomal aminopeptidases on relevant peptides revealed rapid removal of N-terminal Met but not N-terminal Asp. Brain aminopeptidases showed similar specificity. Thus, aminopeptidases would convert Aβ extended by Met into regular Aβ (Asp1) found in amyloid plaques. Earlier studies indicate that Aβ is likely produced in the endosome and lysosome system where cathepsins S, B and L are localized and cysteine cathepsin inhibitors reduce the level of Aβ in cells and animals. Taken together, cathepsins S, B and L deserve further evaluation as therapeutic targets to develop disease modifying drugs to treat Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, it seems important to develop selective inhibitors for human DPPIV (hDPPIV) that are able to control the biological function of hDPPIV. In order to elucidate the binding mode and substrate specificity, we determined the crystal structure complex of hDPPIV and diprotin A (Ile-Pro- Ile), a slowly hydrolyzed substrate of hDPPIV, at 2.2 Å resolution. In this paper, we discuss the molecular interaction mechanism of diprotin A with hDPPIV based on the X-ray crystal structure. [ABSTRACT FROM AUTHOR]

The efficacy of HIV-1 protease (PR) inhibition therapies is often compromised by the emergence of mutations in the PR molecule that reduces the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for natural substrates. In the present study, we used a recombinant HIV-1 C-SA PR and a recently reported variant for inhibition (Ki, IC50) and thermodynamic studies against nine clinically used inhibitors. This is the first time that binding free energies for C-SA PR and the mutant are reported. This variant PR harbours a mutation and insertion (I36T↑T) at position 36 of the C-SA HIV-1 PR, and did not show a significant difference in the catalytic effect of the HIV-1 PR. However, the nine clinically approved HIV PR drugs used in this study demonstrated weaker inhibition and lower binding affinities toward the variant when compared to the wild type HIV-1 PR. All the protease inhibitors (PIs), except Amprenavir and Ritonavir exhibited a significant decrease in binding affinity (p < 0.0001). Darunavir and Nelfinavir exhibited the weakest binding affinity, 155- and 95-fold decreases respectively, toward the variant. Vitality values for the variant PR, against the seven selected PIs, confirm the impact of the mutation and insertion on the South African HIV-1 subtype C PR. This information has important clinical implications for thousands of patients in Sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Kallikrein-related peptidase-3 (KLK3, known also as prostate-specific antigen, PSA) is highly expressed in the prostate. KLK3 possess antiangiogenic activity, which we have found to be related to its proteolytic activity. Thus, it may be possible to slow down the growth of prostatic tumors by enhancing this activity. We have developed peptides that enhance the proteolytic activity of KLK3. As these peptides are degraded in circulation and rapidly excreted, we have started to modify them and have succeeded in creating bioactive and more stable pseudopeptides. We have also identified small molecules stimulating the activity of KLK3, especially in synergy with peptides. [ABSTRACT FROM AUTHOR]

Substance P (SP), an undecapeptide belonging to the tachykinin family, is released during the activation of sensory nerves, and causes vasodilation, edema and pain through activation of tissular Neurokinin 1 receptors. SP proinflammatory effects are terminated by angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), while the aminopeptidase dipeptidylpeptidase IV (DPPIV) can also play a role. The aim of this randomized, crossover, double-blind study was to assess the cutaneous vasoreactivity (flare and wheal reaction, burning pain sensation) to intradermal injection of ascending doses of SP in six volunteers receiving a single therapeutic dose of the DPPIV inhibitor sitagliptin or a matching placebo. Cutaneous SP challenges produced the expected, dose-dependent flare and wheal response, while eliciting mild to moderate local pain sensation with little dose dependency. However, no differences were shown in the responses observed under sitagliptin compared with placebo, while the study would have been sufficiently powered to detect a clinically relevant increase in sensitivity to SP. The results of this pilot study are in line with proteolytic cleavage of SP by ACE and NEP compensating the blockade of DPPIV to prevent an augmentation of its proinflammatory action. [ABSTRACT FROM AUTHOR]

Here we report a detailed analysis of magnesium (Mg2+) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg2+ ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR↓FAGV-Q-EDDnp (from measles virus fusion protein Fo) and Abz-RERRRKKR↓GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl2. It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg2+ is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg2+ ions, which bind to furin with a Kd value of 1.1 m. [ABSTRACT FROM AUTHOR]

Beta-amyloid (Aβ) in the brain is a major factor involved in Alzheimer's disease (AD) that results in severe memory deficit. Our recent studies demonstrate pharmacogenetic differences in the effects of inhibitors of cathepsin B to improve memory and reduce Aβ in different mouse models of AD. The inhibitors improve memory and reduce brain Aβ in mice expressing the wild-type (WT) β-secretase site of human APP, expressed in most AD patients. However, these inhibitors have no effect in mice expressing the rare Swedish (Swe) mutant amyloid precursor protein (APP). Knockout of the cathepsin B decreased brain Aβ in mice expressing WT APP, validating cathepsin B as the target. The specificity of cathepsin B to cleave the WT β-secretase site, but not the Swe mutant site, of APP for Aβ production explains the distinct inhibitor responses in the different AD mouse models. In contrast to cathepsin B, the BACE1 β-secretase prefers to cleave the Swe mutant site. Discussion of BACE1 data in the field indicate that they do not preclude cathepsin B as also being a β-secretase. Cathepsin B and BACE1 could participate jointly as β-secretases. Significantly, the majority of AD patients express WT APP and, therefore, inhibitors of cathepsin B represent candidate drugs for AD. [ABSTRACT FROM AUTHOR]

Proteases can regulate many aspects of tumor development as their actions, which include degradation of the extracellular matrix, proteolytic processing of chemokines and activation of other enzymes, influence several key tumorigenic processes. Members of one protease class, the cysteine cathepsins, have received increasing recognition for their involvement in cancer development, and numerous clinical studies have reported correlations between elevated cathepsin levels and malignant progression. This is also the case for cathepsin H, a member of the cysteine cathepsin family, and its utility as a prognostic marker has been analyzed extensively. However, there is limited information available on its specific functions in tumor development and progression. To gain further insight into the role of this protease in cancer, we crossed cathepsin H-deficient mice with the RIP1-Tag2 model of pancreatic islet carcinogenesis. Deletion of cathepsin H significantly impaired angiogenic switching of the pre-malignant hyperplastic islets and resulted in a reduction in the subsequent number of tumors that formed. Moreover, the tumor burden in cathepsin H null RT2 mice was significantly reduced, in association with defects in the blood vasculature and increased apoptosis. Thus, we demonstrate here for the first time important tumor-promoting roles for cathepsin H in vivo using a mouse model of human cancer. [ABSTRACT FROM AUTHOR]

Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked α/β-heterodimer having a trypsin serine protease-like β-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn2+ metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met β-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation. [ABSTRACT FROM AUTHOR]

Proteases of the serine and cysteine protease families are involved in many processes crucial to the lytic functions of cytotoxic T lymphocytes and natural killer cells. In this study we describe those functions and attempt to place them in the pathophysiological context of defence to pathogen invasion. In particular, we stress that the co-evolution of pathogens with the immune systems of higher organisms over evolutionary time has ensured that redundancy, flexibility and polymorphism of the proteases can be identified, both within the protease repertoire of a given species, and by comparing orthologous protease functions across species. [ABSTRACT FROM AUTHOR]

Proteases play vital roles in a range of biological processes, such as cell cycle, cell growth and differentiation, apoptosis, haemostasis and signalling. Fundamental to our knowledge of protease action is an understanding of how the active site operates; this has been examined through extensive studies of the substrate specificity of the enzymes. Kinetic and structural analyses have shown that the binding of a particular substrate residue at a protease subsite can have either a positive or negative influence on the binding of particular residues at other subsites. This phenomenon has been termed subsite cooperativity and has been observed in a wide range of proteases, often between non-adjacent subsites. This review aims to highlight studies where subsite cooperativity has been observed, experimental techniques used in the past and potential methods that can be employed to comprehensively examine this phenomenon. Further understanding of how the protease active site recognises and chooses its substrates for cleavage will have a significant impact on the development of pharmaceuticals that target these enzymes. [ABSTRACT FROM AUTHOR]

Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans. [ABSTRACT FROM AUTHOR]

Tissue kallikrein KLK1 and the kallikrein-related peptidases KLK2–15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores determined at the time of serological sampling or at last clinical follow-up. Serum levels of KLK1 and KLK6 were elevated in MS patients ( p≤0.027), with highest levels associated with secondary progressive disease. Elevated KLK1 correlated with higher EDSS scores at the time of serum draw and KLK6 with future EDSS worsening in relapsing remitting patients ( p≤0.007). Supporting the concept that KLK1 and KLK6 promote degenerative events associated with progressive MS, exposure of murine cortical neurons to either kallikrein promoted rapid neurite retraction and neuron loss. These novel findings suggest that KLK1 and KLK6 may serve as serological markers of progressive MS and contribute directly to the development of neurological disability by promoting axonal injury and neuron cell death. [ABSTRACT FROM AUTHOR]

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family – a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes. [ABSTRACT FROM AUTHOR]

Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3′. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47–53 and 258–292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition. [ABSTRACT FROM AUTHOR]

Calpains, the cytoplasmic Ca2+-activated regulatory proteases, have no simple and clearly definable cleavage site specificity, which is in sharp contrast to digestive (e.g., pancreatic) proteases. For calpains, an approximate 10-aa segment having a variety of sequences and spanning the scissile bond, governs proteolytic cleavage. This permissivity is a precondition for calpains to act on several different substrate proteins in the cell. The specificity of calpain action may be ensured by anchoring/targeting proteins. Intriguingly, the established endogenous inhibitor protein, calpastatin, might also serve as a storage site. Furthermore, specificity may be encoded in the ‘goodness’ of the undecapeptide sequence in substrate proteins. Novel approaches are needed to reveal how calpains find their substrates in cells at the proper time and location. [ABSTRACT FROM AUTHOR]

The thyroglobulin type-1 (Tg-1) domain is a protein module that occurs in a variety of secreted and membrane proteins and is recognised as a potent inhibitor of cysteine peptidases. We present here some properties of the Tg-1 domain of human testican, a modularly organised proteoglycan secreted mainly by brain cells, the exact in vivo function of which is not yet clear. The domain was prepared as a recombinant protein in a Pichia pastoris expression system and its activity was demonstrated by specific and selective inhibition of cathepsin L ( K i =0.14 nM). Interaction at high enzyme and inhibitor concentrations resulted in degradation of the domain by cathepsin L, which was not observed under conditions used for the determination of kinetic parameters. No inhibitory activity could be detected for cathepsin K, but it exhibited a very similar degradation pattern. Homology modelling provided a good explanation for the different behaviour observed with the two enzymes. Firstly, the steric fit between the interfaces of testican domain and cathepsin L is stabilised by numerous favourable forces, while no such interactions are evident in the complex with cathepsin K, and repulsive interactions even prevent access of the domain to the active site of papain. Secondly, the prolonged first loop of the domain occupies a position near the catalytic cysteine residue in a more substrate-like manner, enabling cleavage of the Gly 22 -Ala 23 bond. [ABSTRACT FROM AUTHOR]

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addi-tion to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or α(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified -- in addition to the C-terminal region of cytokeratin 18 (CK18182-430) -- a large C-terminal fragment of α-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4287-911 and Act-4330-911, respectively, were isolated. An Act-4330-911/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4330-911/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis. [ABSTRACT FROM AUTHOR]

No abstract available [ABSTRACT FROM AUTHOR]

The article discusses the 2nd International Symposium on Kallikreins and Kallikrein-Related Peptidases Symposium. The symposium was held by the Hellenic Anticancer Institute and organized by president Litsa Talieri and Judith Clements, chair of the scientific advisory board. Research on structural features and functional roles of kallikrein was presented by 135 participants. Clements was awarded for her contributions to the identification of the expanded tissue kallikrein gene locus.

No abstract available. [ABSTRACT FROM AUTHOR]

The article discusses kallikreins and kallikrein-related peptidases. The author explains that kallikrein is a proteolytic enzyme that creates Lys-bradykinin for L-kininogen. He states that in 1999 investigators applied molecular biology techniques and discovered that the human kallikrein gene locus contains several other genes similar to KLK1. The article also discusses the different substrate specificities and biological functions of peptidases.

The editorial introduces the present issue containing presentations made at the 4th General Meeting of the International Proteolysis Society in Quebec. Important topics covered at the meeting included research in basic, applied and life sciences, as well as medicine, with added stress on the structure and function relationships of proteases and their inhibitors and the roles that these molecules play in biology and disease.