It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non‐selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X‐ray crystal structure of human α‐thrombin was determined in its complex with the specific thrombin inhibitor d‐Phe‐Pro‐Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467–3475). The crystallographic refinement of the PPACK‐thrombin model has now been completed at an R value of 0.156 (8 to 1.92 Å); in particular, the amino‐ and the carboxy‐termini of the thrombin A‐chain are now defined and all side‐chain atoms localized; only proline 37 was found to be in a cis‐peptidyl conformation.The thrombin B‐chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymo‐trypsin with a root‐mean‐square (r.m.s.) deviation of 0.8 Å for their α‐carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A‐chain is arranged in a boomeranglike shape against the B‐chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding.Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the car‐boxy‐terminal B‐chain helix (forming the presumed heparin‐binding site) and on the surface of loop segment 70–80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A‐chain, in the coherence between the A‐ and the B‐chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67–80).The most remarkable feature at the thrombin surface is the prominent canyonlike active‐site cleft mainly shaped by two characteristic insertion loops around Trp 60D and Trp 148. The deep and narrow active‐site cleft in general explains the narrow specificity of thrombin for distinct macromolecular substrates and inhibitors. Comparisons with other crystal structures of human and bovine thrombin recently determined using this PPACK‐thrombin model indicate that the first loop around Trp 60D is relatively rigid, whereas the opposite loop around Trp 148 can attain different conformations depending on complexation state and crystalline environment.The active‐site residues and the entrance to the specificity pocket are partially occluded in thrombin (much more than in the other serine proteinases) by this distinctive Trp 60D loop. The specificity pocket of thrombin resembles that of bovine trypsin but is designed to prefer arginine over lysine residues at PI. D‐Phe II and Pro 21 of the bound PPACK inhibitor fit neatly to a novel hydrophobic cleft (the aryl‐binding site) and to the cavitylike hydrophobic S2 subsite; the d‐configuration of Phe II is beneficial for binding as it allows the PPACK amino‐terminus to form hydrogen bonds to Gly 216 in addition. Some small arginine and benzamidine‐derived synthetic inhibitors owe their particularly high thrombin specificity and affinity to their exceptional steric fit to these novel hydrophobic cavities close to the thrombin active site (Bode, W., Turk, D., & Stürzebecher, J., 1990, Eur. J. Biochem. 193, 175–182).The active‐site cleft levels off in the primed direction and continues over the molecular surface of the thrombin loop Lys 70‐Glu 80 (itself structurally similar to the calcium loop in trypsin, with the distal nitrogen of Lys 70 replacing the calcium of trypsin). This site of a strong positive electrostatic surface potential probably represents the secondary site for interaction with the α‐chain of fibrinogen and fibrin (the fibrin[ogen] secondary binding exosite) and accommodates the carboxy‐terminal acidic tail part of hirudin (Rydel, T.J., Ravichandran, K.G., Tulinsky, A., Bode, W., Huber, R., Roitsch, C., & Fenton, J.W., II, 1990, Science 249, 277–280). Segment Arg 187‐Gly 188‐Asp 189, which could represent a thrombin adhesion site for cellular interactions with platelets, fibroblasts, and endothelial cells, is mainly buried in α‐thrombin; its adhesive role would thus appear to require some prior unfolding.Most of the well‐characterized sites of proteolytic cleavage leading to the degradation products β‐, γ‐, and ϵ‐thrombin of diminished or lost clotting activity are situated in exposed mobile loops of α‐thrombin. None of these segments is in a canonical conformation that would allow association with the substrate‐binding site of a cleaving serine proteinase without large conformational changes. The cleavage of the Arg 77A‐Asn 78 scissile peptide bond (leading to β‐thrombin) presumably results in the unfolding of the 70–80 loop, exposing the salt bridge‐connected residues buried in α‐thrombin to the solvent; the concomitant disruption of the surface of the fibrin(ogen) secondary binding exosite would explain the loss of binding capacity (and thus catalytic activity) toward fibrinogen. The Arg 67‐Ile 68 peptide bond is completely buried beneath this 70–80 loop surface and thus only susceptible to an attacking proteinase after prior cleavage and exposure of this loop, as observed.

A large-scale screen to target SARS-CoV-2: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, M ^pro , is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to M ^pro . High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science , this issue p. 642

The cystatin superfamily comprises a large group of the cystatin domain containing proteins, present in a wide variety of organisms, including humans. Cystatin inhibitory activity is vital for the delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases such as the papain (C1) family, including cysteine cathepsins. Some of the cystatins also inhibit the legumain (C13) family of enzymes. Failures in biological mechanisms controlling protease activities result in many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis, arthritis, and cancer. Cystatins have been classified into three types: the stefins, the cystatins and the kininogens, although other cystatin-related proteins, such as CRES proteins, are emerging. The stefins are mainly intracellular proteins, whereas the cystatins and the kininogens are extracellular. The cystatins are tight binding and reversible inhibitors. The basic mechanism of interaction between cystatins and their target proteases has been established, based mainly on the crystal structures of various cathepsins, stefins and cystatins and their enzyme-inhibitor complexes. Cystatins, as rather non-selective inhibitors, discriminate only slightly between endo- and exopeptidases. They are also prone to form amyloids. The levels of some stefins and cystatins in tissue and body fluids can serve as relatively reliable markers for a variety of diseases. In this review we summarize present knowledge about cystatins and their role in some diseases.