Evidence for Restricted Reactivity of ADAMDEC1 with Protein Substrates and Endogenous Inhibitors

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1′ position. Cys³⁹², present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys³⁹² for a Ser increased the reactivity with α₂-macroglobulin but not with casein or Cm-Tf. Substitution of Asp³⁶² for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.     

ADAMTS-12 Metalloprotease Is Necessary for Normal Inflammatory Response

ADAMTSs (a disintegrin and metalloprotease with thrombospondin domains) are a family of enzymes with both proteolytic and protein interaction functions, which have been implicated in distinct pathologies. In this work, we have investigated the putative role of ADAMTS-12 in inflammation by using a mouse model deficient in this metalloprotease. Control and mutant mice were subjected to different experimental conditions to induce colitis, endotoxic sepsis, and pancreatitis. We have observed that Adamts12-deficient mice exhibit more severe inflammation and a delayed recovery from these challenges compared with their wild-type littermates. These changes are accompanied by an increase in inflammatory markers including several cytokines, as assessed by microarray expression analysis and proteomic-based approaches. Interestingly, the clinical symptoms observed in Adamts12-deficient mice are also concomitant with an elevation in the number of neutrophils in affected tissues. Finally, isolation and in vitro culture of human neutrophils demonstrate that the presence of ADAMTS-12 induces neutrophil apoptosis. On the basis of these results, we propose that ADAMTS-12 is implicated in the inflammatory response by modulating normal neutrophil apoptosis.     

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.     

ADAM10 Is the Major Sheddase Responsible for the Release of Membrane-associated Meprin A

Meprin A, composed of α and β subunits, is a membrane-bound metalloproteinase in renal proximal tubules. Meprin A plays an important role in tubular epithelial cell injury during acute kidney injury (AKI). The present study demonstrated that during ischemia-reperfusion-induced AKI, meprin A was shed from proximal tubule membranes, as evident from its redistribution toward the basolateral side, proteolytic processing in the membranes, and excretion in the urine. To identify the proteolytic enzyme responsible for shedding of meprin A, we generated stable HEK cell lines expressing meprin β alone and both meprin α and meprin β for the expression of meprin A. Phorbol 12-myristate 13-acetate and ionomycin stimulated ectodomain shedding of meprin β and meprin A. Among the inhibitors of various proteases, the broad spectrum inhibitor of the ADAM family of proteases, tumor necrosis factor-α protease inhibitor (TAPI-1), was most effective in preventing constitutive, phorbol 12-myristate 13-acetate-, and ionomycin-stimulated shedding of meprin β and meprin A in the medium of both transfectants. The use of differential inhibitors for ADAM10 and ADAM17 indicated that ADAM10 inhibition is sufficient to block shedding. In agreement with these results, small interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited meprin β and meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhanced shedding of meprin β from both transfectants. Our studies demonstrate that ADAM10 is the major ADAM metalloproteinase responsible for the constitutive and stimulated shedding of meprin β and meprin A. These studies further suggest that inhibiting ADAM 10 activity could be of therapeutic benefit in AKI.     

A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg²⁺ binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.     

Activity of ADAM17 (a Disintegrin and Metalloprotease 17) Is Regulated by Its Noncatalytic Domains and Secondary Structure of its Substrates

ADAM proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off target toxicity. Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strategy for drug discovery; however, there are only a few reports of potential exosites in ADAM protease structures. In the study presented here, we utilized a series of TNFα-based substrates to probe ADAM10 and 17 interactions with its canonical substrate to identify the structural features that determine ADAM protease substrate specificity. We found that noncatalytic domains of ADAM17 did not directly bind the substrates used in the study but affected the binding nevertheless, most likely because of steric hindrance. Additionally, noncatalytic domains of ADAM17 affected the size/shape of the carbohydrate-binding pocket contained within the catalytic domain of ADAM17. This suggests that noncatalytic domains of ADAM17 play a role in substrate specificity and might help explain differences in substrate repertoires of ADAM17 and its closest homologue, ADAM10. We also addressed the question of which substrate features can affect ADAM protease specificity. We found that all ADAM proteases tested (i.e., ADAM10, 12, and 17) significantly decreased activity when the TNFα-derived sequence was induced into α-helical conformation, suggesting that conformation plays a role in determining ADAM protease substrate specificity. These findings can help in the discovery of ADAM isoform- and substrate-specific inhibitors.     

Structural and functional analyses reveal that Staphylococcus aureus antibiotic resistance factor HmrA is a zinc-dependent endopeptidase

HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or β-lactamase but a nonspecific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface. Four HmrA protomers assemble to a large ∼170-kDa homotetrameric complex of 125 Å. All four active sites are fully accessible and ∼50–70 Å apart, far enough apart to act on a large meshwork substrate independently but simultaneously. In vivo studies with four S. aureus strains of variable resistance levels revealed that the extracellular addition of HmrA protects against loss of viability in the presence of oxacillin and that this protection depends on proteolytic activity. All of these results indicate that HmrA is a peptidase that participates in resistance mechanisms in vivo in the presence of β-lactams. Furthermore, our results have implications for most S. aureus strains of known genomic sequences and several other cocci and bacilli, which harbor close orthologs. This suggests that HmrA may be a new widespread antibiotic resistance factor in bacteria.     

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

Protocadherin-12 cleavage is a regulated process mediated by ADAM10 protein: evidence of shedding up-regulation in pre-eclampsia

Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E(2), or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity.     

Fungal recognition enhances mannose receptor shedding through dectin-1 engagement

The mannose receptor (MR) is an endocytic type I membrane molecule with a broad ligand specificity that is involved in both hemostasis and pathogen recognition. Membrane-anchored MR is cleaved by a metalloproteinase into functional soluble MR (sMR) composed of the extracellular domains of intact MR. Although sMR production was initially considered a constitutive process, enhanced MR shedding has been observed in response to the fungal pathogen Pneumocystis carinii. In this work, we have investigated the mechanism mediating enhanced MR shedding in response to fungi. We show that other fungal species, including Candida albicans and Aspergillus fumigatus, together with zymosan, a preparation of the cell wall of Saccharomyces cerevisiae, mimic the effect of P. carinii on sMR production and that this effect takes place mainly through β-glucan recognition. Additionally, we demonstrate that MR cleavage in response to C. albicans and bioactive particulate β-glucan requires expression of dectin-1. Our data, obtained using specific inhibitors, are consistent with the canonical Syk-mediated pathway triggered by dectin-1 being mainly responsible for inducing MR shedding, with Raf-1 being partially involved. As in the case of steady-state conditions, MR shedding in response to C. albicans and β-glucan particles requires metalloprotease activity. The induction of MR shedding by dectin-1 has clear implications for the role of MR in fungal recognition, as sMR was previously shown to retain the ability to bind fungal pathogens and can interact with numerous host molecules, including lysosomal hydrolases. Thus, MR cleavage could also impact on the magnitude of inflammation during fungal infection.     

Proteolytic processing of dentin sialophosphoprotein (DSPP) is essential to dentinogenesis

DSPP, which plays a crucial role in dentin formation, is processed into the NH(2)-terminal and COOH-terminal fragments. We believe that the proteolytic processing of DSPP is an essential activation step for its biological function in biomineralization. We tested this hypothesis by analyzing transgenic mice expressing the mutant D452A-DSPP in the Dspp-knock-out (Dspp-KO) background (referred to as "Dspp-KO/D452A-Tg" mice). We employed multipronged approaches to characterize the dentin of the Dspp-KO/D452A-Tg mice, in comparison with Dspp-KO mice and mice expressing the normal DSPP transgene in the Dspp-KO background (named Dspp-KO/normal-Tg mice). Our analyses showed that 90% of the D452A-DSPP in the dentin of Dspp-KO/D452A-Tg mice was not cleaved, indicating that D452A substitution effectively blocked the proteolytic processing of DSPP in vivo. While the expression of the normal DSPP fully rescued the dentin defects of the Dspp-KO mice, expressing the D452A-DSPP failed to do so. These results indicate that the proteolytic processing of DSPP is an activation step essential to its biological function in dentinogenesis.     

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys⁴⁷ of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys¹⁰¹, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys¹⁰¹-NO is substantially more resistant to denitrosation than Cys⁴⁷-NO, suggesting a potential role for Cys¹⁰¹ in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.     

Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis

Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular factors. Here, we show that sumoylation promotes EV71 3C protein ubiquitination for degradation, correlating with a decrease of EV71 in virus replication and cell apoptosis. SUMO E2-conjugating enzyme Ubc9 was identified as an EV71 3C-interacting protein. Further studies revealed that EV71 3C can be SUMO (small ubiquitin-like modifier)-modified at residue Lys-52. Sumoylation down-regulated 3C protease activity in vitro and also 3C protein stability in cells, in agreement with data suggesting 3C K52R protein induced greater substrate cleavage and apoptosis in cells. More importantly, the recombinant EV71 3C K52R virus infection conferred more apoptotic phenotype and increased virus levels in culture cells, which also correlated with a mouse model showing increased levels of viral VP1 protein in intestine and neuron loss in the spinal cord with EV71 3C K52R recombinant viral infection. Finally, we show that EV71 3C amino acid residues 45-52 involved in Ubc9 interaction determined the extent of 3C sumoylation and protein stability. Our results uncover a previously undescribed cellular regulatory event against EV71 virus replication and host cell apoptosis by sumoylation at 3C protease.     

Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.     

N-glycoform diversity of cellobiohydrolase I from Penicillium decumbens and synergism of nonhydrolytic glycoform in cellulose degradation

Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.