Mcm10 Self-Association Is Mediated by an N-Terminal Coiled-Coil Domain

Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.     

Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

Ectodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase Cδ (PKCδ). Downregulation of PKCδ inhibits neuregulin release and reduces ex vivo neurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate.     

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.     

Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer.     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.     

Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain

Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe viruses, which are not known to naturally cause human diseases. Using a recombinant Pichinde virus system, we show that the LCMV Z N-terminal domain (NTD) mediates the inhibition of macrophage activation and immune functions.     

PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P₃ phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call “hopping.” Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.     

Cleavage Mediated by the Catalytic Domain of Bacterial RNase P RNA

Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.     

Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca²⁺-activated ion channel and Ca²⁺-activated phospholipid scramblase activities, requiring a high intracellular Ca²⁺ concentration (e.g., half-maximal effective Ca²⁺ concentration [EC₅₀] of [Ca²⁺]_i > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca²⁺-activated chloride channel exhibiting higher Ca²⁺ sensitivity (EC₅₀ of 1 μM) than ANO6, suggested that a homologous Ca²⁺-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca²⁺ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca²⁺-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6-1-6 showed higher sensitivity to Ca²⁺ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca²⁺ interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca²⁺-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca²⁺ sensitivity, implying direct interactions of Ca²⁺ with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca²⁺ sensitivity between ANO1 and ANO6.     

Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1

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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.     

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.     

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.     

Cleavage of neuregulin-1 by BACE1 or ADAM10 protein produces differential effects on myelination

Neuregulin-1 (Nrg1) is encoded by a single gene and exists in naturally secreted and transmembrane isoforms. Nrg1 exerts its signaling activity through interaction with its cognate ErbB receptors. Multiple membrane-anchored Nrg1 isoforms, present in six different membrane topologies, must be processed by a protease to initiate a signaling cascade. Here, we demonstrate that BACE1 and ADAM10 can process type I and III Nrg1 at two adjacent sites. Our cleavage site mapping experiments showed that the BACE1 cleavage site is located eight amino acids downstream of the ADAM10 cleavage site, and this order of cleavage is the opposite of amyloid precursor protein cleavage by these two enzymes. Cleavages were further confirmed via optimized electrophoresis. Cleavage of type I or III Nrg1 by ADAM10 and BACE1 released a signaling-capable N-terminal fragment (ntf), either Nrg1-ntfα or Nrg1-ntfβ, which could similarly activate an ErbB receptor as evidenced by increased phosphorylation of Akt and ERK, two downstream signaling molecules. Although both Nrg1-ntfα and Nrg1-ntfβ could initiate a common signaling cascade, inhibition or down-regulation of ADAM10 alone in a co-culture system did not affect normal myelination, whereas specific inhibition of BACE1 impaired normal myelination. Thus, processing of Nrg1 by BACE1 appears to be more critical for regulating myelination. Our results imply that a significant inhibition of BACE1 could potentially impair Nrg1 signaling activity in vivo.     

Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation. In addition, siRNA to Unc93b1 reduced the secretion of the TLR3 ectodomain (T3ECD) into the cell medium. Furthermore, two human single nucleotide polymorphisms that affected herpesvirus and influenza virus encephalopathy as well as a natural isoform generated by alternative splicing were found to be impaired for T3ECD secretion and decreased the abundance of TLR3 on the cell surface. The locations of the SNP P554S and the deletion in the isoform led to the identification of a loop in the TLR3 ectodomain that is required for secretion and a second whose presence decreased secretion. Finally, a truncated protein containing the N-terminal 10 leucine-rich repeats of T3ECD was sufficient for secretion in an Unc93b1-dependent manner.     

The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na⁺ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.Amiloride-sensitive epithelial Na⁺ channels (ENaC)³ are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1–4). Na⁺ absorption via ENaC is critical to the normal regulation of Na⁺ and fluid homeostasis and is important for maintaining blood pressure (5) and the volume of fluid in the respiratory passages (6). Increased ENaC activity has been implicated in the salt-sensitive inherited form of hypertension, Liddle''s syndrome (7), and dehydration of the surface of the airway epithelium in the pathology associated with cystic fibrosis lung disease (8).Expression of ENaC at the cell membrane surface is regulated by the E3 ubiquitin protein ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) (9). Interaction between the WW domains of Nedd4-2 and the proline-rich PY motifs (PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect on the channel (10, 11). This interaction leads to ubiquitination-dependent internalization of ENaC (12, 13). Several regulators of ENaC exert their effects on the channel by modulating the action of Nedd4-2. For instance, serum and glucocorticoid-dependent protein kinase (14), protein kinase B (15), and G protein-coupled receptor kinase (16) up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of cellular mechanisms that underlie internalization of ENaC remain to be elucidated, the physiological significance of Nedd4-dependent internalization of the channel has been well established. For instance, heritable mutations that delete the cytosolic termini of the β-or γ-subunit of ENaC, which contain the proline-rich motifs, are known to cause hyperactivity of ENaC in the kidney (17) and increase cell surface expression of the channel (7, 18).The plasma membranes of most cell types contain lipid raft microdomains that are enriched with glycosphingolipid and cholesterol (19), that have distinctive biophysical properties, and that selectively include or exclude signaling molecules (20). These microdomains promote clustering of an array of integral membrane proteins in the membrane leaflets (21) and may be important for organizing cascades of signaling molecules (22, 23). Processes in which raft microdomains are involved include the intracellular transport of proteins and lipids to the cell membrane (24), the endocytotic retrieval of membrane proteins (25, 26), and signal transduction (27, 28). In addition, segregation of signaling molecules within lipid rafts may facilitate cross-talk between signal transduction pathways (29), a phenomenon that may be important in ensuring rapid and efficient integration of multiple cellular signaling events (30, 31). Of particular interest is the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1 (Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been identified as being involved in diverse cellular functions, such as vesicular transport, cholesterol homeostasis, and signal transduction (32). Cav-1 also regulates the activity and membrane expression of ion channels and transporters (28).In epithelia, the majority of lipid rafts exist at the apical membrane surface (22). Pools of ENaC (33–36) and several proteins that regulate activity of ENaC, such as Nedd4 (37), protein kinase B (38), protein kinase C (39), G_o (40), and the G protein-coupled receptor kinase (41), have been identified in detergent-insoluble and cholesterol-rich membrane fractions from a variety of cell types, consistent with localization of these proteins in lipid rafts. Furthermore, detergent-free buoyant density separation of lipid rafts has revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane fraction (35). The physiological role of lipid rafts in the regulation of ENaC has been the subject of many recent investigations. Most of these studies used a pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote redistribution of proteins away from the cholesterol-enriched membrane domains. The results were, however, inconclusive. In some studies, MβCD treatment was found to inhibit open probability (42) or cell surface expression of ENaC (35), whereas others found no direct effect of MβCD on the channel (33, 43).Despite a number of studies into the role of lipid rafts on the regulation of ENaC, little is known about the physiological relevance of caveolins to the function of this ion channel. In the present study, we use gene interference and gene expression techniques to determine the role of Cav-1 in the regulation of ENaC activity. We provide evidence of the association of Cav-1 with ENaC and evidence that Cav-1 negatively regulates both activity and abundance of ENaC at the surface of epithelial cells. Importantly, we demonstrate, for the first time, that the mechanism by which Cav-1 regulates activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2.