Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

Ubiquitination is one of the most crucial post-translational protein modifications involved in a myriad of biological pathways. This process is reversed by deubiquitinases (DUBs) that deconjugate the ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors and have previously successfully applied it to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the tight JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of the STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates the UbV technology is suitable to develop tool molecules for metalloproteases. These tools can be used to understand the cellular function of JAMM family DUBs.

Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach

Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado–Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0–135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Deubiquitinating Enzyme Amino Acid Profiling Reveals a Class of Ubiquitin Esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome and many have been implicated with pathologies including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct and its physiological importance is emerging. Here we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain (OTU) DUB class demonstrate specific (iso)peptidase activity. Strikingly, we find the Machado Josephin Domain (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity that rivals the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad but proximal hydrophobic residues appear to be general determinants of non-lysine activity. These findings suggest that non-lysine ubiquitination is an integral component of the ubiquitin system, with regulatory sophistication comparable to that of canonical ubiquitination.

Role of the COP9 Signalosome (CSN) in Cardiovascular Diseases

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is an evolutionarily conserved multi-protein complex, consisting of eight subunits termed CSN1-CSN8. The main biochemical function of the CSN is the control of protein degradation via the ubiquitin-proteasome-system through regulation of cullin-RING E3-ligase (CRL) activity by deNEDDylation of cullins, but the CSN also serves as a docking platform for signaling proteins. The catalytic deNEDDylase (isopeptidase) activity of the complex is executed by CSN5, but only efficiently occurs in the three-dimensional architectural context of the complex. Due to its positioning in a central cellular pathway connected to cell responses such as cell-cycle, proliferation, and signaling, the CSN has been implicated in several human diseases, with most evidence available for a role in cancer. However, emerging evidence also suggests that the CSN is involved in inflammation and cardiovascular diseases. This is both due to its role in controlling CRLs, regulating components of key inflammatory pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and complex-independent interactions of subunits such as CSN5 with inflammatory proteins. In this case, we summarize and discuss studies suggesting that the CSN may have a key role in cardiovascular diseases such as atherosclerosis and heart failure. We discuss the implicated molecular mechanisms ranging from inflammatory NF-κB signaling to proteotoxicity and necrosis, covering disease-relevant cell types such as myeloid and endothelial cells or cardiomyocytes. While the CSN is considered to be disease-exacerbating in most cancer entities, the cardiovascular studies suggest potent protective activities in the vasculature and heart. The underlying mechanisms and potential therapeutic avenues will be critically discussed.