The last years have seen a steady increase in our understanding of legumain biology that is driven from two largely uncoupled research arenas, the mammalian and the plant legumain field. Research on ...legumain, which is also referred to as asparaginyl endopeptidase (AEP) or vacuolar processing enzyme (VPE), is slivered, however. Here we summarise recent important findings and put them into a common perspective. Legumain is usually associated with its cysteine endopeptidase activity in lysosomes where it contributes to antigen processing for class II MHC presentation. However, newly recognized functions disperse previously assumed boundaries with respect to their cellular compartmentalisation and enzymatic activities. Legumain is also found extracellularly and even translocates to the cytosol and the nucleus, with seemingly incompatible pH and redox potential. These different milieus translate into changes of legumain's molecular properties, including its (auto-)activation, conformational stability and enzymatic functions. Contrasting its endopeptidase activity, legumain can develop a carboxypeptidase activity which remains stable at neutral pH. Moreover, legumain features a peptide ligase activity, with intriguing mechanistic peculiarities in plant and human isoforms. In pathological settings, such as cancer or Alzheimer's disease, the proper association of legumain activities with the corresponding cellular compartments is breached. Legumain's increasingly recognized physiological and pathological roles also indicate future research opportunities in this vibrant field.
•Legumain can have endopeptidase, carboxypeptidase or ligase activity.•Legumain activities are milieu (pH, redox potential, cofactor, substrate) dependent.•Legumain can localize to the endo/lysosome, cell surface, cytosol or nucleus.•Breached compartmentalization of legumain activities reflects the disease state.
The cysteine protease legumain plays important functions in immunity and cancer at different cellular locations, some of which appeared conflicting with its proteolytic activity and stability. Here, ...we report crystal structures of legumain in the zymogenic and fully activated form in complex with different substrate analogs. We show that the eponymous asparagine-specific endopeptidase activity is electrostatically generated by pH shift. Completely unexpectedly, the structure points toward a hidden carboxypeptidase activity that develops upon proteolytic activation with the release of an activation peptide. These activation routes reconcile the enigmatic pH stability of legumain, e.g., lysosomal, nuclear, and extracellular activities with relevance in immunology and cancer. Substrate access and turnover is controlled by selective protonation of the S1 pocket (K M) and the catalytic nucleophile (k cₐₜ), respectively. The multibranched and context-dependent activation process of legumain illustrates how proteases can act not only as signal transducers but as decision makers.
Legumain is a lysosomal cysteine protease with strict specificity for cleaving after asparagine residues. By sequence comparison, legumain belongs to MEROPS clan CD of the cysteine proteases, which ...indicates its structural and mechanistic relation to caspases. Contrasting caspases, legumain harbors a pH-dependent ligase activity in addition to the protease activity. Although we already have a significant body of knowledge on the catalytic activities of legumain, many mechanistic details are still elusive. In this study, we provide evidence that extended active site residues and substrate conformation are steering legumain activities. Biochemical experiments and bioinformatics analysis showed that the catalytic Cys189 and His148 residues are regulated by sterically close Glu190, Ser215 and Asn42 residues. While Glu190 serves as an activity brake, Ser215 and Asn42 have a favorable effect on legumain protease activity. Mutagenesis studies using caspase-9 as model enzyme additionally showed that a similar Glu190 activity brake is also implemented in the caspases. Furthermore, we show that the substrate’s conformational flexibility determines whether it will be hydrolyzed or ligated by legumain. The functional understanding of the extended active site residues and of substrate prerequisites will allow us to engineer proteases with increased enzymatic activity and better ligase substrates, with relevance for biotechnological applications.
Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at ...least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn2+ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α2-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.
δ-secretase, also known as asparagine endopeptidase (AEP) or legumain, is a lysosomal cysteine protease that cleaves both amyloid precursor protein (APP) and tau, mediating the amyloid-β and tau ...pathology in Alzheimer's disease (AD). Here we report the therapeutic effect of an orally bioactive and brain permeable δ-secretase inhibitor in mouse models of AD. We performed a high-throughput screen and identified a non-toxic and selective δ-secretase inhibitor, termed compound 11, that specifically blocks δ-secretase but not other related cysteine proteases. Co-crystal structure analysis revealed a dual active site-directed and allosteric inhibition mode of this compound class. Chronic treatment of tau P301S and 5XFAD transgenic mice with this inhibitor reduces tau and APP cleavage, ameliorates synapse loss and augments long-term potentiation, resulting in protection of memory. Therefore, these findings demonstrate that this δ-secretase inhibitor may be an effective clinical therapeutic agent towards AD.
Collagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid composition and ...triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochemical and enzymatic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degradation assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.
The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v ...1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.
Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.
The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.
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► Ligand binding to Bet v 1 may contribute to explain its allergenicity. ► High-resolution structures reveal the binding mode of diverse ligands to Bet v 1. ► Residue 30 starkly influences the binding properties of different Bet v 1 isoforms. ► Ternary complexes with diverse ligands explain anomalous fluorescence binding curves. ► Betv1 isoforms differ in ligand binding, which may translate into their allergenicity.
Collagen constitutes one-third of body protein in humans, reflecting its extensive role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that have evolved for ...collagen remodeling. The most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues; but despite intense study, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55-Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which recognition and unraveling of collagen microfibrils into triple helices, as well as unwinding of the triple helices, are driven by collagenase opening and closing.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background
Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic ...sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen–derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity.
Methods
We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T‐cell epitope presentation in BMDCs.
Results
We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen‐derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity.
Conclusion
Bet v 1 can serve as a transporter of pollen‐derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen‐centered view to a more systemic view that includes the host endolysosomal enzymes.
The newly identified birch pollen‐derived ligands, Phytoprostane E1 (PPE1), interacts with Bet v 1 affecting its stability and proteolytic processing. Ubiquitous plant phytoprostanes, PPE1, covalently inhibit lysosomal cysteine cathepsins, with multiple consequences including effects on antigen processing. PPE1 affected the presentation of Bet v 1 T‐cell epitope in BMDC to T‐cell.
Human coagulation factor IX serves both to maintain and to control blood coagulation. The dual function of this hemophilic factor is implemented by a tiered activation mechanism. Processed two-chain ...factor IXa is catalytically silent; only together with its cofactor VIIIa does factor IXa form the highly potent Xase complex. The detailed mechanism of this secondary activation has remained elusive so far. Here we present the crystal structures of Xase-like factor IXa mutants with several-thousand-fold activity enhancement that mimic the secondary activation by Xase formation. The structures reveal how cofactor-triggered and substrate-assisted modulations in the factor IXa 99- and 60-loops cooperate in S4 through S2′ formation, allowing for productive substrate recognition. We could further physically map and visualize a distinct communication line, along which agonists such as Ca2+ direct their effects to the active site and vice versa.
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