C-terminal Cleavage Fragment Research Articles

A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans

Abstract A20 is an NF-κB-induced, dual function ubiquitin editing enzyme that negatively regulates NF-κB signaling induced by TNF and other innate immune receptors, constraining inflammation. Indeed, A20 haploinsufficiency drives a severe autoinflammatory disease in humans. Although A20 serves a similar negative feedback function for T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun. We employed an extensive set of mutant expression constructs to better elucidate how A20 regulates TCR signaling output in Jurkat T cells. Intriguingly, A20 knockout cells showed enhanced, sustained NF-κB and JNK signaling after stimulation, which was reversed upon wild-type (WT) A20 complementation. A20 deubiquitinase function was entirely dispensable for NF-κB and JNK regulation, whereas zinc finger domains that confer E3 ligase activity (ZnF4) and linear ubiquitin-binding capacity (ZnF7) were required. Abolishing MALT1-dependent proteolytic cleavage of A20 did not affect suppressive function, although neither N- nor C-terminal A20 cleavage fragments retained regulatory capacity. We also found that CRISPR-mediated deletion of A20 adversely affects the survival and expansion of primary human T cells. Ongoing studies will determine how A20 deletion affects human T cell effector responses after stimulation, and decipher how dysregulated NF-κB and/or JNK signaling contributes to T cell-intrinsic abnormalities that perturb adaptive immune homeostasis.

Structure‒function‒pathogenicity analysis of C-terminal myocilin missense variants based on experiments and 3D models.

MYOC is a common pathogenic gene for primary open-angle glaucoma and encodes the protein named myocilin. Multiple MYOC variations have been found, with different clinical significance. However, the pathogenesis of glaucoma induced by MYOC mutations has not been fully clarified. Here, we analyze the molecular and cellular biological differences caused by multiple variant myocilins, including protein secretion characteristics, structural changes, subcellular localization, cellular autophagic activity and oxidative stress. Denaturing and nondenaturing electrophoresis showed myocilin to be a secreted protein with the tendency to self-oligomerize. The full-length myocilin and its C-terminal cleavage fragment are secreted. Secretion analysis of 23 variant myocilins indicated that secretion defects are closely related to the pathogenicity of MYOC variants. Structural analysis showed that the alteration of steric clash is associated with the secretion characteristics and pathogenicity of myocilin variants. Immunocytochemistry results demonstrated that mutated myocilins are retained in the endoplasmic reticulum and disrupt autophagy. MTT assay, MitoTracker staining, and DCFH-DA staining showed increased oxidative injury in cells expressing MYOC mutants. Taken together, MYOC mutations are able to induce cell dysfunction via secretion defects and intracellular accumulation resulting from steric clash alterations.

RIN4 homologs from important crop species differentially regulate the Arabidopsis NB-LRR immune receptor, RPS2

RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.

MALT-1 inhibition attenuates the inflammatory response of ankylosing spondylitis by targeting NF-κB activation

The present study aimed to investigate the effects of mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 on ankylosing spondylitis and its underlying mechanisms. Proteoglycan induced ankylosing spondylitis (PGIA) mouse model was established and the expression patterns of MALT-1 were determined in joint tissue. Next, the mice were intraarticularly administrated with MALT-1 in the PGIA mouse model. Meanwhile, shRNA was intraarticularly administrated to PGIA mice. The incidence of arthritis and clinical score was evaluated. Besides, the levels of inflammatory cytokines and matrix metalloproteinases (MMPs) were measured. Protein expressions of full-length CYLD (FL-CYLD), C-terminal cleavage fragment (CYLD-CL), and nuclear factor (NF)-κB were determined. The mRNA and protein levels of MALT1 were increased in the PGIA mouse model. The treatment of MALT-1 accelerated arthritis incidence and joint damage, whereas shMALT-1 suppressed arthritis symptoms in the PGIA mouse model. In addition, treatment of shMALT-1 suppressed the levels of inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β), MMP-3, and MMP-9. Furthermore, the treatment of shMALT-1 suppressed the levels of CYLD and NF-κB in the joint tissues in the PGIA mouse model. The inhibition of MALT-1 suppressed the inflammatory response in ankylosing spondylitis in part by the regulation of CYLD and NF-κB.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.

MP77-10 URINARY AGRIN CLEAVAGE PRODUCTS PROVIDE PHENOTYPIC CHARACTERIZATION OF INTERSTITIAL CYSTITIS PATIENTS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Non-neurogenic Voiding Dysfunction II1 Apr 2016MP77-10 URINARY AGRIN CLEAVAGE PRODUCTS PROVIDE PHENOTYPIC CHARACTERIZATION OF INTERSTITIAL CYSTITIS PATIENTS Laura Lamb, Peter Levanovich, Andrew Vereecke, Bernadette Zwaans, Sarah Bartolone, Kenneth Peters, and Michael Chancellor Laura LambLaura Lamb More articles by this author , Peter LevanovichPeter Levanovich More articles by this author , Andrew VereeckeAndrew Vereecke More articles by this author , Bernadette ZwaansBernadette Zwaans More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , Kenneth PetersKenneth Peters More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1924AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Many patients with interstitial cystitis/bladder pain syndrome (IC/BPS) have pelvic floor muscle tenderness and may also have pelvic floor disorder. This can contribute to poor urination stream, need to push or bear down when voiding, painful intercourse, urinary urgency and frequency issues, pelvic pain, and incomplete bladder emptying. Agrin is involved in the organization of acetylcholine receptor clustering and plays a pivotal role in the formation and maintenance of neuromuscular junctions (NMJ), the cholinergic connections between nerves and muscles. When agrin is cleaved by neurotrypsin into two C-terminal cleavage fragments, there is a loss in the stabilization of the NMJ. Alterations in agrin expression results in loss of muscle coordination and is associated with neuromuscular diseases such as Amyotrophic Lateral Sclerosis and Sarcopenia. We sought to determine if agrin levels were altered in IC/BPS patients. METHODS Midstream urinary specimens were collected from 13 control and 17 female nonulcerative IC/BPS patients. IC/BPS patients had IC/BPS diagnosis for a minimum of 6 months and a total IC Symptom and Problem Indices (ICSI/PI) score greater than 16 (24.4±4.5). Samples were age and gender matched. Total LG3 containing C-terminal fragments were measured by enzyme-linked immunosorbent assay. Data was analyzed using Prism (GraphPad) software. RESULTS Total C-terminal fragments of agrin in urine samples were 137.4±28.13 pM in IC/BPS patients compared to 29.71±11.63 pM controls, resulting in a 4.62 fold increase (p=0.0014). Total agrin fragments also correlated with pain (p=0.0237) and frequency symptom scores (p=0.0373) of the ICSI. CONCLUSIONS Agrin plays an important role in the formation, maintenance, and reinforcement of NMJs, and IC/BPS patients have elevated urine levels of cleaved agrin. These results indicate that agrin depletion at the NMJ may be an important event in the development of pelvic floor dysfunction and pelvic pain in IC/BPS patients. Thus, agrin fragment detection in urine might be of diagnostic value. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1019-e1020 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Laura Lamb More articles by this author Peter Levanovich More articles by this author Andrew Vereecke More articles by this author Bernadette Zwaans More articles by this author Sarah Bartolone More articles by this author Kenneth Peters More articles by this author Michael Chancellor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone

The utility of serum anti-Müllerian hormone (AMH) assays in assessment of female fertility have been investigated extensively but little is known about the biological activity of the hormone being studied. ProAMH is the proprotein precursor and is incapable of binding to the AMH-specific type II receptor. Proteolytic cleavage generates receptor-competent AMHN,C which is a non-covalent complex of the N- and C-terminal cleavage fragments. Commercially available AMH assays do not differentiate the two forms of AMH. Techniques were developed to dissociate the AMHN,C complex and abolish its two-site immunoassay immunoreactivity. This allowed specific quantification of proAMH. The surfactant sodium deoxycholate (DOC) dissociated AMHN,C without disrupting binding of proAMH to the capture-antibody with an optimal concentration of 0.1–0.2%w/v. The incorporation of a DOC incubation step into the AMH Gen II ELISA detected proAMH, with AMHN,C cross-detection conservatively estimated at 6.0% ± 2.5% (mean ± S.D.). The intra-assay and inter-assay variability were estimated at 8.0%CV and 13.0%CV respectively. The levels of proAMH and total AMH were assessed in 5 boys and 5 men and the proportion of proAMH was found to be significantly higher in boys (p = 0.005). This study will facilitate further investigation of the role of proteolytic cleavage in AMH signalling.

Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A-mediated C-terminal dystrophin cleavage fragment.

Enterovirus infection can cause severe cardiomyopathy in humans. The virus-encoded 2A protease is known to cleave the cytoskeletal protein dystrophin. It is unclear, however, whether cardiomyopathy results from the loss of dystrophin or is due to the emergence of a dominant-negative dystrophin cleavage product. We show for the first time that the 2A protease-mediated carboxyl-terminal dystrophin cleavage fragment (CtermDys) is sufficient to cause marked dystrophic cardiomyopathy. The sarcolemma-localized CtermDys fragment caused myocardial fibrosis, heightened susceptibility to myocardial ischemic injury, and increased mortality during cardiac stress testing in vivo. CtermDys cardiomyopathy was more severe than in hearts completely lacking dystrophin. In vivo titration of CtermDys peptide content revealed an inverse relationship between the decay of membrane-bound CtermDys and the restoration of full-length dystrophin at the sarcolemma, in support of a physiologically relevant loss of dystrophin function in this model. CtermDys gene titration and dystrophin replacement studies further established a target threshold of 50% membrane-bound intact dystrophin necessary to prevent mice from CtermDys cardiomyopathy. Conversely, the NtermDys fragment did not compete with dystrophin and had no pathological effect. Thus, CtermDys must be localized to the sarcolemma, with intact dystrophin <50% of normal levels, to exert dominant-negative peptide-dependent cardiomyopathy. These data support a two-hit dominant-negative disease mechanism where membrane-associated CtermDys severs the link to cortical actin and inhibits both full-length dystrophin and compensatory utrophin from binding at the membrane. Therefore, membrane-bound CtermDys is a new potential translational target for virus-mediated cardiomyopathy.

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis.

We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at amino-acid 327 between glutamine and alanine, resulting in the generation of an N- and C-terminal cleavage fragment of ~35 and ~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection.

Cleavage of the JunB Transcription Factor by Caspases Generates a Carboxyl-terminal Fragment That Inhibits Activator Protein-1 Transcriptional Activity

The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription.

Increased expression and cleavage of amyloid precursor-like protein 2 in pancreatic cancer and regulation of fragment stability by major histocompatibility complex class I molecules (165.4)

Abstract Particularly in neurological systems, amyloid precursor-like protein 2 (APLP2) has been linked to cellular growth, adhesion and migration. Our laboratory has demonstrated that several human pancreatic cancer cell lines, as well as pancreatic cancer tissue, express a high level of APLP2. Furthermore, we showed that oncogene-induced transformation of pancreatic cells resulted in increased levels of full-length APLP2 and 12-15 kDa APLP2 C-terminal cleavage fragments. Additionally, in pancreatic cancer cell lines we have noted association of APLP2 C-terminal fragments and major histocompatibility complex (MHC) class I molecules, the proteins responsible for the presentation of tumor antigens. The APLP2 C-terminal fragments associated with MHC class I molecules had prolonged stability compared to unassociated fragments. Incubation with calyculin A, which enhances serine/threonine phosphorylation, increased the rate of APLP2 C-terminus turnover. In agreement with our earlier finding, the APLP2 C-termini associated with MHC class I molecules underwent relatively slower degradation in the presence of calyculin A. Thus, MHC class I molecules have a previously unrecognized function of regulating APLP2 homeostasis. Overall, our discoveries have established that APLP2 expression and cleavage are up-regulated in pancreatic cancer, and have led to a new understanding of the involvement of MHC class I molecules in the regulation of APLP2 C-terminal fragments in pancreatic cancer cells.

International Union of Basic and Clinical Pharmacology. LXXVII. Kisspeptin Receptor Nomenclature, Distribution, and Function

Kisspeptins are members of the Arg-Phe amide family of peptides, which have been identified as endogenous ligands for a G-protein-coupled receptor encoded by a gene originally called GPR54 (also known as AXOR12 or hOT7T175). After this pairing, the gene has been renamed KISS1R. The International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification recommends that the official name for the receptor is the kisspeptin receptor to follow the convention of naming the receptor protein after the endogenous ligand. The endogenous ligand was initially called metastin, after its role as a metastasis suppressor, and is now referred to as kisspeptin-54 (KP-54), a C-terminally amidated 54-amino acid peptide cleaved from the 145-amino acid gene product. Shorter C-terminal cleavage fragments [KP-14, KP-13 and KP-10 (the smallest active fragment)] are also biologically active. Both receptor and peptide are widely expressed in human, rat, and mouse; the receptor sequence shares more than 80% homology in these species. Activation of the kisspeptin receptor by kisspeptin is via coupling to G(q/11) and the phospholipase C pathway, causing Ca(2+) mobilization. Mutations in the KISS1R gene result in hypogonadotropic hypogonadotropism, and targeted disruption of Kiss1r in mice reproduces this phenotype, which led to the discovery of the remarkable ability of the kisspeptin receptor to act as a molecular switch for puberty. In addition to regulating the reproductive axis, the kisspeptin receptor is also implicated in cancer, placentation, diabetes, and the cardiovascular system.

ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by γ-secretase

Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using γ-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the γ-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After γ-secretase cleavage IL6R cell surface expression was lost and γ-secretase cleavage product(s) of the IL6R were endocytosed. No GFP-fluorescence of a γ-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded.

Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active.

Fibroblast growth factor 23 (FGF23) regulates phosphorus homeostasis and vitamin D metabolism. Circulating FGF23 levels are elevated in inherited and acquired hypophosphatemic disorders that can cause rickets or osteomalacia. Particularly increased concentrations of FGF23 are observed in patients with chronic kidney disease (CKD), in which increased FGF23 is associated with more rapid disease progression, improved bone mineralization, the development of left ventricular hypertrophy, and increased mortality. Our objective was to determine whether the markedly elevated levels of immunoreactive FGF23 in CKD represent accumulation of intact, biologically active hormone, C-terminal cleavage fragments, or both. Biologically active FGF23 in plasma from CKD patients treated by peritoneal dialysis was quantified using a cell-based Egr-1 reporter assay; bioactive FGF23 levels were compared with those measured with immunometric FGF23 assays detecting either intact hormone alone or intact hormone and C-terminal fragments. Adult and pediatric patients with end-stage renal disease treated with peritoneal dialysis participated in the study at a tertiary referral center. Serially diluted patient samples revealed levels of bioactive FGF23 that ran in parallel to CHO cell-derived recombinant human FGF23. FGF23 bioactivity was inhibited by an anti-FGF23 antibody. Levels of bioactive and immunoreactive FGF23 were tightly correlated, and Western blot analysis of FGF23 immunoprecipitated with anti-FGF23 antibodies from plasma of dialysis patients revealed only a single prominent protein band, which was indistinguishable from recombinant intact FGF23, without clear evidence for FGF23 fragments. Our results provide strong evidence for the conclusion that virtually all circulating FGF23 in dialysis patients is intact and biologically active.

Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53

Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53(-/-) mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.

Nedd4-2 Induces Endocytosis and Degradation of Proteolytically Cleaved Epithelial Na+ Channels

As a pathway for Na(+) reabsorption, the epithelial Na(+) channel ENaC is critical for Na(+) homeostasis and blood pressure control. Na(+) transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved alphaENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.

Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.

Cleavage in the GDPH sequence of the C-terminal cysteine-rich part of the human MUC5AC mucin

MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Identification of Proteins Cleaved Downstream of Caspase Activation in Monocytes Undergoing Macrophage Differentiation

We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.