Inactive Protease Research Articles

Circulating Extracellular Vesicle Tissue Factor Activity in Chuvash Erythrocytosis

Background:Chuvash erythrocytosis (CE), an inherited autosomal recessive disease endemic to Russia's mid-Volga River region, is caused by a germlineVHLC598T mutation (encoding VHLR200W) that alters oxygen sensing (PMID: 12415268). VHLR200W protein displays impaired degradation of hypoxia inducible transcription factor (HIF)-α subunits leading to increased HIF-1 and HIF-2 in normoxia and increased transcription of many HIF-regulated genes including erythropoietin (PMID: 14726398). CE patients have increased risk of venous and arterial thromboses, which are the major cause of morbidity and mortality (PMID:28104701). Thrombosis occurs despite lower blood pressure, body mass index and white blood cells compared to controls and is not related to the elevation in hematocrit but is increased in patients treated with phlebotomy therapy (PMID: 31289208). We have shown by microarray analysis of CE peripheral blood mononuclear cells modestly increased expression of several HIF-regulated pro-thrombotic genes at false discovery rate <0.05, includingIL1B(encoding interleukin-1beta),THBS1(thrombospondin-1),EGR1(early growth response 1),NLRP3,ITGA2B(integrin alpha-IIb),SERPINE1(plasminogen activator inhibitor-1) andF3(tissue factor) (PMID: 23993337). TF is a primary initiator of coagulation that binds factor VII/VIIa. The TF/factor VIIa complex catalyzes the conversion of inactive protease factor X to active protease factor Xa, leading to thrombin and thrombus formation (PMID: 19923557). EV-TF is barely detectable under basal circumstances but in disease states such as cardiovascular disease, sickle cell disease and cancer it derives from monocytes, endothelial cells, vascular smooth muscle cells and tumor cells (PMIDs: 20690821, 12805058, 17166244, 14988149, 26916302). Granulocytes of patients with polycythemia vera and essential thrombocytopenia expressF3as assessed by RT-qPCR (PMID: 32203583). Because of the central role of tissue factor in initiating the extrinsic pathway of coagulation, we set up to determine if extracellular vesicle associated tissue factor activity (EV-TF) could be detected in the plasma of CE patients. Methods:EightVHLC598T homozygotes and 6VHLwild type controls from Cheboksary, Chuvashia, Russia were studied as outpatients under basal circumstances. Blood was collected from these participants by venipuncture into vacutainer tubes containing 0.129 M sodium citrate. Immediately after venipuncture, platelet free plasma (PFP) was prepared by two rounds of centrifugation at 2,500 × g for 15 minutes at room temperature and EVs were isolated from PFP by centrifugation at 20,000 g for 15 minutes at 4 ºC. EV-TF was measured in duplicate by a two-stage Factor Xa generation assay with and without anti-TF antibody using Innovin (Siemens Healthcare Diagnostics) as a standard (PMID: 30656275). We also isolated granulocyte mRNA from 3 of the patients and 2 of the controls at a different time point and measured F3 transcripts by RT-qPCR. Results:We detected EV-TF in 4 of 8 CE patients but in no wild type controls (one-sided P = 0.043). The range of 0.45 to 1.25 pg/ml is similar to the range recently reported in US patients with cancer (PMID: 32548563). MCHC tended to be lower among 4 CE patients with detectable EV-TF than 4 without (one-sided P = 0.12), but this was not the case for serum ferritin. We also detectedF3transcripts in granulocytes, and these levels were higher in the 3 patients than the 2 controls that we analyzed (one-sided P = 0.12). Furthermore,F3mRNA correlated with plasma EV-TF in these five subjects (Spearman rho = 0.71, one-sided P < 0.05). As previously reported, CE patients had higher hemoglobin, hematocrit and red blood cells and lower platelets compared to controls (PMID: 14726398). They also had lower values for serum ferritin and mean corpuscular hemoglobin concentration (MCHC) suggesting iron deficiency, likely induced by phlebotomy therapy or by the presence of gastritis, which is increased in CE. Discussion:The presence of EV-TF in the plasma of 4 out of 8 CE patients and none of six controls, and increasedF3transcripts in CE granulocytes, may point to a potential thrombogenic role, although none of the CE patients in this study had a history of thrombosis. Further studies on larger numbers of patients are warranted to confirm these findings and to clarify the potential role of EV-TF in thrombosis in CE. *NM, JTP & VRG contributed equally Figure Disclosures Gordeuk: CSL Behring:Consultancy, Research Funding;Ironwood:Research Funding;Imara:Research Funding;Global Blood Therapeutics:Consultancy, Research Funding;Novartis:Consultancy.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B’/B and C’/C. POST1 promotes the generation of the functional S1P-C’/C from S1P-B’/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in Bacillus subtilis.

Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in Bacillus subtilis utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from S.pyogenes) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in B.subtilis has a 5 nt editing window, which is similar to previously reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in B.subtilis. Finally, we applied multiplex genome editing to generate a B.subtilis 168 mutant strain with eight inactive extracellular protease genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in B.subtilis and industrial applications in the future.

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites.

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparumendoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

Structure, dynamics and immunogenicity of a catalytically inactive CXC chemokine-degrading protease SpyCEP from Streptococcus pyogenes

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

X-ray structure of an inactive zymogen clostripain-like protease from Parabacteroides distasonis.

The clostripain-like (C11) family of cysteine proteases are ubiquitously produced by the vast majority of the bacterial strains that make up the human distal gut microbiome. Recent reports show that some C11 proteases promote host immune responses and bacterial pathogenesis, including the induction of neutrophil phagocytosis and the activation of bacterial pathogenic toxins, respectively. The crystal structure of distapain, the only C11 protease predicted within the genome of the commensal bacterium Parabacteroides distasonis, was determined in the inactive zymogen state to 1.65 Å resolution. This is the first C11 protease structure of a zymogen, and the structure helped to uncover key unique conformations among critical active-site residues that are likely to assist in preserving the inactive protease. His135, a member of the catalytic dyad, is repositioned approximately 5.5 Å from the orientation found in active C11 structures and forms a hydrogen bond to Asp180 and a π-stacking interaction with Trp133. The structure sheds light on the potential importance of Asp180 and Trp133, as these residues are highly conserved across C11 proteases. Structure elucidation of C11 proteases will ultimately help to identify new ways to chemically and/or biologically regulate this family of enzymes, which represent potential drug-discovery targets in microbiome-related gastrointestinal diseases.

Phylogenetic relationships, stage-specific expression and localisation of a unique family of inactive cysteine proteases in Sarcoptes scabiei

BackgroundScabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites. We postulated that the SMIPP-Cs have evolved as an adaptation to the parasitic lifestyle of the scabies mite. To formulate testable hypotheses for their functions and to propose possible strategies for translational research we investigated whether the SMIPP-Cs are common to all scabies mite varieties and where within the mite body as well as when throughout the parasitic life-cycle they are expressed.ResultsSMIPP-C sequences from human, pig and dog mites were analysed bioinformatically and the phylogenetic relationships between the SMIPP-C multi-copy gene families of human, pig and dog mites were established. Results suggest that amplification of the SMIPP-C genes occurred in a common ancestor and individual genes evolved independently in the different mite varieties. Recombinant human mite SMIPP-C proteins were produced and used for murine polyclonal antibody production. Immunohistology on skin sections from human patients localised the SMIPP-Cs in the mite gut and in mite faeces within in the epidermal skin burrows. SMIPP-C transcription into mRNA in different life stages was assessed in human and pig mites by reverse transcription followed by droplet digital PCR (ddPCR). High transcription levels of SMIPP-C genes were detected in the adult female life stage in comparison to all other life stages.ConclusionsThe fact that the SMIPP-Cs are unique to three Sarcoptes varieties, present in all burrowing life stages and highly expressed in the digestive system of the infective adult female life stage may highlight an essential role in parasitism. As they are excreted from the gut in scybala they presumably are able to interact or interfere with host proteins present in the epidermis.

ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.

Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent

Coagulation factor XIa is a candidate target for anticoagulants that better separate antithrombotic efficacy from bleeding risk. We report a co-crystal structure of the FXIa protease domain with DEF, a human monoclonal antibody that blocks FXIa function and prevents thrombosis in animal models without detectable increased bleeding. The light chain of DEF occludes the FXIa S1 subsite and active site, while the heavy chain provides electrostatic interactions with the surface of FXIa. The structure accounts for the specificity of DEF for FXIa over its zymogen and related proteases, its active-site-dependent binding, and its ability to inhibit substrate cleavage. The inactive FXIa protease domain used to obtain the DEF-FXIa crystal structure reversed anticoagulant activity of DEF in plasma and invivo and the activity of a small-molecule FXIa active-site inhibitor invitro. DEF and this reversal agent for FXIa active-site inhibitors may help support clinical development of FXIa-targeting anticoagulants.

Next-Generation Trapping of Protease Substrates by Label-Free Proteomics.

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug‐resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size‐exclusion chromatography, we obtained from HCV‐seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low‐density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma‐derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine‐binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851‐1864)

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2−/− mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this ‘stress’ keratin is regulated.

Characterizing Dynamic Protein-Protein Interactions Using the Genetically Encoded Split Biosensor Assay Technique Split TEV.

Dynamic protein-protein interactions (PPIs) are fundamental building blocks of cellular signaling and monitoring their regulation promotes the understanding of signaling in health and disease. Genetically encoded split protein biosensor assays, such as the split TEV method, have proved to be highly valuable when studying regulated PPIs in living cells. Split TEV is based on the functional complementation of two previously inactive TEV protease fragments fused to interacting proteins and provides a robust, sensitive and flexible readout to monitor PPIs both at the membrane and in the cytosol. Thus, split TEV can be used to analyze interactomes of receptors, membrane-associated proteins, and cytosolic proteins. In particular, split TEV is useful to assay activities of relevant drug targets, such as receptor tyrosine kinases and G protein-coupled receptors, in compound screens. As split TEV uses genetically encoded readouts, including standard reporters based on fluorescence and luminescence, the technique can also be combined with scalable molecular barcode reporter systems, allowing the integration into multiplexed high-throughput assay approaches. Split TEV can be used in standard heterologous cell lines and primary cell types, including neurons, either in a transient or stably integrated format. When using cell lines, the basic protocol takes 30-96 h to complete, depending on the complexity of the experimental question addressed.

The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9.

The secretory proprotein convertase (PC) family comprises nine members, as follows: PC1/3, PC2, furin, PC4, PC5/6, paired basic amino acid cleaving enzyme 4, PC7, subtilisin kexin isozyme 1/site 1 protease (SKI-1/S1P), and PC subtilisin/kexin type 9 (PCSK9). The first seven PCs cleave their substrates at single/paired basic residues and exhibit specific and often essential functions during development and/or in adulthood. The essential SKI-1/S1P cleaves membrane-bound transcription factors at nonbasic residues. In contrast, PCSK9 cleaves itself once, and the secreted inactive protease drags the low-density lipoprotein receptors (LDLR) and very LDLR (VLDLR) to endosomal/lysosomal degradation. Inhibitory PCSK9 monoclonal antibodies are now prescribed to treat hypercholesterolemia. This review focuses on the implication of PCs in cardiovascular functions and diseases, with a major emphasis on PCSK9. We present a phylogeny of the PCs and the analysis of PCSK9 haplotypes in modern and archaic human species. The absence of PCSK9 in mice led to the discovery of a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR. PCSK9 inhibition may have other applications because it reduces inflammation and sepsis in a LDLR-dependent manner. Our present understanding of the cellular mechanism(s) that enables PCSK9 to induce the degradation of receptors is reviewed, as well as the consequences of its key natural mutations. The PCSK9 ongoing clinical trials are reviewed. Finally, how the other PCs may impact cardiovascular disease and the metabolic syndrome, and become relevant targets, is discussed.

The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding.

HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release.

Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex

The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichiacoli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus.

Recent studies have defined several virulence factors as vaccine candidates against Vibrio vulnificus. However, most of these factors have the potential to cause pathogenic effects in the vaccinees or induce incomplete protection. To overcome these drawbacks, a catalytically inactive form, CPDVv (C3725S), of the well-conserved cysteine protease domain (CPD) of V. vulnificus multifunctional autoprocessing repeats-in-toxin (MARTXVv /RtxA1) was recombinantly generated and characterized. Notably, active and passive immunization with CPDVv (C3725S) conferred protective immunity against V. vulnificus strains. These results may provide a novel framework for developing safe and efficient subunit vaccines and/or therapeutics against V. vulnificus that target the CPD of MARTX toxins.

Cleavage-Dependent and Independent Role of the Serine Protease CAP1/Prss8

Serine protease are enzymes involved in the regulation of many biological processes, and generally exert their function by cleaving peptide bonds through the catalytic triad of histidine, aspartate and serine. The channel-activating protease 1 (CAP1/protease serine S1 family member 8 (Prss8) is a membrane-anchored serine protease expressed in the epithelium of several organs, such as the skin, colon, lung and kidney, and can be released in the extracellular space by the action of phospholipase C. CAP1/Prss8 was one of the the first of several membrane-bound serine proteases, such as CAP2/Tmptrss4 and CAP3/matriptase, found to activate the epithelial sodium channel (ENaC) in-vitro. Since these studies strongly suggested a non-enzymatic function of CAP1/Prss8, we over-expressed the catalytically inactive form of CAP1/Prss8 and asked whether we can negate the obtained phenotype by crossing these mice with mice lacking the protease-activated receptor 2, that we previously identified as downstream target substrate. Since serine protease activity needs to be tighly regulated, we also asked the question whether the putative serine protease inhibitor nexin-1 is able to block CAP1/Prss8 in vitro and in vivo.Our results demonstrate the ability of a catalytically inactive serine protease to induce disease when ectopically expressed, and a novel inhibitory interaction that does not depend on its catalytic site.

Ligand Binding to Anion Binding Exosites as Prothrombin is Converted to Active Thrombin

The blood coagulant thrombin is originally expressed as the zymogen prothrombin (ProT). Prothrombin contains the F1.2 (Gla + Kringle 1 + Kringle 2) domain and the inactive serine protease prethrombin-2 (PT2). Kringle 2 of F1.2 protects an immature anion binding exosite on ProT called pro-ABE II. An immature, but unprotected, pro-ABE I site also exists on ProT. As part of the zymogen activation process, the prothrombinase complex can cleave ProT after R271 and release F1.2 from PT2. Subsequent cleavage of PT2 at R320 helps to generate active thrombin. Both anion binding exosites become fully mature upon production of thrombin. In the current work, solution NMR studies have been used to characterize the binding of GpIbα (269-286, 15N-labeled L275, 15N-labeled D277) to (pro)-ABE II and the binding of PAR3 (44-56, 15N-labeled F47, 15N-labeled D54 ) to (pro)-ABE I. The residues that are 15N-labeled have previously been shown by X-ray crystallography to make extensive contact with the thrombin surface. This observation is further supported by 1D proton NMR line broadening studies on the ligand-thrombin complexes. 15N-1H HSQC NMR titrations demonstrate that GpIbα (269-286, 15N-labeled L275, 15N-labeled D277) does not bind to the pro-ABE II site on human ProT. Ligand binding affinity to ABE II then increases as ProT is converted to PT2 and then thrombin. By contrast, HSQC NMR titrations reveal that the pro-ABE I site on ProT can already bind PAR3 (44-56, 15N-labeled F47, 15N-labeled D54). More selective therapeutics may take advantage of this immature ABE I site. Binding affinity for PAR3 (44-56) further increases as active thrombin is produced. After the two mature exosites are generated, thrombin is well poised to regulate several coagulation and anti-coagulation events.