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.

SummaryExtracellular signals are often transduced by dynamic signaling complexes (“signalosomes”) assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of β-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to β-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

The subcellular distribution of basic fibroblastic growth factor (bFGF) was analyzed by subcellular fractionation and immunofluorescence to gain insight into potential mechanisms for its release from cells. Subcellular fractionation of either SK-Hep-1 cells or NIH 3T3 cells transfected with a bFGF cDNA revealed that the 18 kd form of bFGF was found primarily in the cytosolic fraction, whereas the 22 and 24 kd forms of bFGF were found preferentially in ribosomal and nuclear fractions. Analysis of bFGF distribution by immunofluorescence using an antibody that recognized all forms of bFGF indicated both cytoplasmic and nuclear localization but failed to reveal any growth factor in structures representing secretory vesicles. Therefore, bFGF has a distribution inconsistent with that of a secretory protein.

Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-[4,5'-bithiazole]-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.

Summary Thyroglobulin is the protein precursor of thyroid hormones, which are essential for growth, development and control of metabolism in vertebrates 1 , 2 . Hormone synthesis from thyroglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis 3 . Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding 4 . Here we present the structure of full-length human thyroglobulin at ~3.5 Å resolution determined by electron cryomicroscopy (cryo-EM). We identified all hormonogenic tyrosine pairs in the structure and verified them via site-directed mutagenesis and in vitro hormone production assays using human TG expressed in HEK cells. Analysis revealed that proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. Transferring the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose binding protein (MBP) yielded hormone production with efficiency comparable to TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

A new trick for an old dog! Aberrant cathepsin B activity is associated with tumor progression, however, despite extensive research, there are no cathepsin B inhibitors in clinical use. Here, nitroxoline, an established antimicrobial agent, is identified as a potent, reversible inhibitor of cathepsin B, and is thus a potential drug candidate for the treatment of cancer and other diseases in which cathepsin B activity plays a role.

Background: Lectins are carbohydrate-binding proteins that exert their activity by binding to specific glycoreceptors. Results: Clitocybe nebularis lectin (CNL) showed biological activity, although its nonsugar-binding and monovalent mutants were inactive. Conclusion:The bivalent carbohydrate-binding property of CNL is essential for its activity. Significance: Understanding the interactions of lectins with glycans and elucidating their modes of action are necessary for their application in biomedicine.

Cysteine cathepsins are involved in protein degradation (1) and the development and function of the immune system (2). Cathepsin L is an endopeptidase that is able to perform limited proteolysis in the endosomes and lysosomes of specific cell types. Besides its role in hair formation and skin metabolism, it is involved in T-cell selection and NKT cell development (3). It participates in processing the major histocompatibility complex II invariant chain in thymic cortex epithelial cells (4), encephalin in chromaffin granules of neuroendocrine cells (5), and in the degradation and recycling of growth factors and their receptors in epidermal keratinocytes (6). Cathepsin L is also associated with an endosomal processing step during invasion of cells by Ebola virus (7), severe acute respiratory syndrome (SARS) coronavirus (8), and murine hepatitis coronavirus (9). As the result of gene duplication, the human genome encodes for two cathepsin L-like proteases, namely the human cathepsin L and cathepsin V (cathepsin L2), whereas in mouse only cathepsin L is present (10). At the protein level, mouse cathepsin L displays a higher sequence homology to human cathepsin V than to human cathepsin L (11). Cathepsin V shares 80% protein sequence identity with cathepsin L, but in contrast to the ubiquitously expressed cathepsin L, its expression is restricted to thymus and testis (11,12).Recently, the otherwise endosomal proteinase cathepsin L has been reported to be active in the nucleus. It cleaves the CUX1 transcription factor and as a result accelerates progression into the S phase of the cell cycle (13). Cathepsin L deficiency was shown to cause a global rearrangement of chromatin structure and redistribution of specifically modified histones (14). In addition, cathepsin L was found to cleave histone H3.2 in the nucleus during mouse embryonic stem cell differentiation (15).Cathepsin L is inhibited in vitro by a number of proteins as follows: cystatins (16), thyropins (17), and some of the serpins (18,19). Type 1 cystatins, or stefins, are mainly intracellular, whereas type 2 cystatins are predominantly secreted (20,21). Stefin B is localized in the cytosol and nucleus of proliferating cells (22). Loss-of-function mutations in the cystatin B (CTSB, stefin B) gene are found in patients with Unverricht-Lundborg disease (EPM1), but its physiological implication in the pathogenesis of the disease has yet to be defined (23-26). EPM1 is an autosomal recessive inherited disease in which patients suffer from myoclonic jerks, tonic-clonic epileptic seizures, and progressive decline in cognition (26). Histopathological examination of the brain has shown neural degeneration in several areas of the central nervous system, with cerebellar damage and serious alterations of Purkinje cells (27). The most common mutation in EPM1 patients is a dodecamer repeat expansion in the stefin B (CSTB) gene promoter region that leads to reduced mRNA and protein levels (23,25). In addition, four mutations in the coding region were reported in EPM1 (23,28...