Abstract Cysteine cathepsins are receiving large attention due to their widespread participation in physiological and pathological processes. The link between their specificity and biological function is more complex than previously anticipated. Structural analysis revealed that this complex specificity is anchored in the architecture of the active site cleft. Unlike classical proteases with well-defined specificity pockets S1, the corresponding P1 residue of a cathepsin substrate points toward solvent, precluding a clear P1-driven specificity profile. This review analyzes the evolution of cysteine cathepsin research, spanning from the initial biochemical era and the “golden age” of structural biology to contemporary high-throughput profiling, degradomics, and computational analysis. The surge in available data has reshaped our understanding, shifting from rigid active-site models toward statistically driven patterns that reflect the inherent flexibility of structure. For cysteine cathepsins, the next frontier lies in discerning redundant versus unique physiological and pathological roles. This requires integrating high-resolution localization profiles, adequate prediction models, and in situ measurements of degradation products. Such integration will mark a new era of proteolysis studies, replacing individual enzyme-centric research with a multidimensional synthesis of diverse data sources encompassing the expanding landscape of proteases and their substrates.
Abstract Crystallographic studies of proteases and inhibitors by Wolfram Bode and colleagues in the late twentieth century relied on simple graphical modeling, force-field energy functions, and human intuition. These systems now provide interesting tests of modern AI methods. We examine three classes of protease inhibitors and their complexes. For cystatin, originally modeled with papain by manual fitting of protein surfaces, AI methods recover both the inhibitor fold and binding geometry to differing extents, with some approaches favoring a single binding mode and others generating broader ensembles that may explore alternative geometries. De novo design against papain reveals a similar contrast between conservative and exploratory modes of prediction. For the two-headed serine protease inhibitor from mung bean, prediction of its symmetric ternary complex with two trypsin molecules depends strongly on recovery of the small disulfide-rich inhibitor fold. Finally, modeling α1-antitrypsin tests whether AI methods can distinguish known uncleaved and cleaved conformations of a structurally more plastic inhibitor. Local restructuring of the central β-sheet is central to mechanism, while the overall fold remains conserved. These applications show diverse uses of AI modeling tools, from reproducing known structures to exploring assemblies and generating novel interactions, while exposing limitations in flexibility and sampling.
Abstract PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are second messengers and key regulators of tumorigenesis. Their production and degradation are spatiotemporally regulated by a network of phosphoinositide kinases and phosphatases which is frequently altered in cancer. Both molecules serve as docking and activation sites for PH-domain containing effector proteins regulating oncogenic traits. This review will detail the proteins and pathways involved in the upstream regulation and downstream functions of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 . In addition, it will cover the spatiotemporal distribution of these two PtdIns and relevant analysis techniques.
Immune cells undergo metabolic reprogramming in response to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (ACOD1), which generates itaconate from cis-aconitate in the TCA cycle. Itaconate inhibits succinate dehydrogenase, resulting in succinate accumulation. Stable ACOD1 overexpression in RAW264.7 cells shifted cellular metabolism towards glycolysis, as indicated by enhanced mTOR activation, increased 4E-BP1 phosphorylation, and reduced ATP levels. ACOD1 cells displayed impaired osteoclastogenesis with reduced expression of osteoclast-associated genes and fewer TRAP-positive multinucleated osteoclasts. Unexpectedly, NFATc1 was constitutively present in the nucleus of untreated ACOD1 cells, resulting in residual NFAT activity and induction of inflammatory genes. Upon RANKL stimulation, these pre-activated cells showed delayed osteoclastogenic signalling accompanied by sustained expression of the transcriptional repressors BCL6, MafB, and IRF8. Using a GPR91 antagonist and a Gαq inhibitor, we demonstrate that extracellular succinate activates NFATc1 via GPR91-Gαq signalling. RNA sequencing further revealed that ACOD1 overexpression promotes an innate immune transcriptional program rather than osteoclast differentiation. Together, our findings identify succinate-GPR91 signalling as a regulator of the transition between inflammatory activation and osteoclastogenesis.
The X-ray crystal structure of astacin from the freshwater crayfish Astacus astacus published in 1992 was the second structural prototype of a zinc metalloendopeptidase solved after thermolysin from Bacillus thermoproteolyticus 20 years earlier. Astacin became the founding member of a clan of metalloendopeptidases, for which Wolfram Bode coined the designation 'metzincins'. This review recapitulates the discovery of the metzincin clan, which includes the astacins, MMPs (matrix metalloproteinases), serralysins and adamalysins/ADAMs (A disintegrin and metalloproteinase enzymes). Currently, over a dozen subclans (families) have been assigned including the pappalysins, leishmanolysins, snapalysins, igalysins, helicolysins, and toxylysins.
