C-terminal Cleavage Product Research Articles

Ebolavirus Δ-Peptide Immunoadhesins Inhibit Marburgvirus and Ebolavirus Cell Entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and "cuevaviruses") cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP(1,2), to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP(1,2), ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP(1,2)-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP(1,2), as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP(1,2)-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.

Abstract LB-134: C-terminal PKCδ1 protein cleavage product acts as a tumor suppressor in human non-small cell lung carcinoma cells

Abstract PKCδ1 is a member of family proteins of the lipid-activated serine/threonine kinases that have been implicated in a wide range of cellular functions, including apoptosis and cell proliferation. While the function of PKCδ1 in regulating apoptosis still remains controversial, it has been well documented that PKCδ1 can undergoe apoptosis-dependent protein cleavage. However, the function of its protein cleavage product has not been previously characterized. The PKCδ1 protein cleavage has also been frequently observed in our studies during apoptosis induced by various apoptosis stimuli, including proteasome inhibitors. To investigate the involvement of PKCδ1 protein cleavage product in apoptosis, in this study, we cloned 3′ portion of PKCδ1 cDNA, which encodes the predicted C-terminal PKCδ1 protein cleavage fragment starting from 318 asparagine residue to the stop codon of the protein, into pcDNA3.1Zeo(+) vector with Flag tag at its 5′end. The new vector was then named Flag-PKCδ1-Δ318. Subsequently, the Flag-PKCδ1-Δ318 demonstrated its ability to express a 35kd protein in COS7 cells following gene transfection. In human non-small cell lung carcinoma (NSCLC) NCI-H1703 and NCI-H358 cells, we observed that PKCδ1 underwent protein cleavage resulting in a 35kd cleavage product during proteasome inhibitor MG132-induced apoptosis. It was also observed that silencing of PKCδ1 expression by its siRNA mildly enhanced apoptosis induction in both NSCLC cell lines, suggesting that PKCδ1 can minimally inhibit MG132-induced apoptosis in NSCLC cells. However, unexpectedly, transfection with Flag-PKCδ1-Δ318, did not affect MG132-induced apoptosis. Instead, it caused a significant inhibition of cell proliferation as demonstrated by the slowed cell growth and reduced colony formation in both NSCLC cell lines. Moreover, it was found that expression of PKCδ1-Δ318 dramatically reduced in vitro migration and abolished in vivo tumor formation of NCI-H358 cells, strongly suggesting that PKCδ1-Δ318 acts as tumor suppressor. To further investigate the possible mechanisms underlying the tumor suppression function of PKCδ1-Δ318, we tested activity of MAPK, AKT and MLCK, the critical kinases associated with many oncogenic properties of tumor cells. It was then found that the activity of all these kinases were significantly reduced by PKCδ1-Δ318 in NCI-H358 cells, suggesting that the PKCδ1-Δ318 protein, different from its parental protein, exerts its tumor suppression function through inhibiting multiple oncogenic signaling pathways. Therefore, this study suggests that the apoptosis-dependent protein cleavage may represent a new mechanism in regulating tumorigenic activities and PKCδ1-Δ318 may be of great value of being a new cancer gene therapy agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-134. doi:10.1158/1538-7445.AM2011-LB-134

MEPE Activated by Furin Promotes Pulpal Cell Adhesion

Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in osteoblasts, osteocytes, and odontoblasts and plays key biological roles in bone and dentin metabolism. Post-translational modifications are essential for its activation. This study tested the hypothesis that MEPE is activated through proteolytic processing by furin in dental pulp. MEPE was present in three sizes, 1 full-length and 2 cleaved fragments; the cleavage site was 146R↓147. The proprotein convertase family, particularly furin, was a candidate enzyme. Introducing a substitution at the cleavage site inhibited hydrolysis, but there was no cleavage of MEPE expressed in furin-deficient LoVo cells. Therefore, furin is a strong candidate for the proteolytic cleavage of MEPE. The C-terminal cleavage product promoted cell adhesion via its RGD motif. These results indicate that proteolytic processing by furin may activate MEPE during its secretion from odontoblasts and may play important roles in dentinogenesis and pulpal homeostasis. Abbreviations: MEPE, matrix extracellular phosphoglycoprotein; PTM, post-translational modifications; OLC, odontoblast-lineage cells.

Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA*

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43-kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300-400-kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes but retained FUS/TLS binding activity. The functional significance of TDP-43-FUS/TLS complexes was established by showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression of histone deacetylase (HDAC) 6 mRNA. TDP-43 and FUS/TLS associated with HDAC6 mRNA in intact cells and in vitro, and competition experiments suggested that the proteins occupy overlapping binding sites. The combined findings demonstrate that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may reflect their participation in common biochemical processes.

Cleavage of transaldolase by granzyme B causes the loss of enzymatic activity with retention of antigenicity for multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8(+) CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2-restricted TAL 168-176-specific CD8(+) T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28-337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8(+) T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.

1178 THE EMERGING ROLE OF PROGRP IN PROSTATE CARCINOGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research VI1 Apr 20101178 THE EMERGING ROLE OF PROGRP IN PROSTATE CARCINOGENESIS Chew-Lin Yip, Rachele Lockie, Joseph Ischia, Liesl Ischia, Oneel Patel, Damien Bolton, Graham Baldwin, and Arthur Schulkes Chew-Lin YipChew-Lin Yip Heidelberg, Melbourne, Australia More articles by this author , Rachele LockieRachele Lockie Heidelberg, Melbourne, Australia More articles by this author , Joseph IschiaJoseph Ischia Heidelberg, Melbourne, Australia More articles by this author , Liesl IschiaLiesl Ischia Heidelberg, Melbourne, Australia More articles by this author , Oneel PatelOneel Patel Heidelberg, Melbourne, Australia More articles by this author , Damien BoltonDamien Bolton Melbourne, Australia More articles by this author , Graham BaldwinGraham Baldwin Heidelberg, Melbourne, Australia More articles by this author , and Arthur SchulkesArthur Schulkes Heidelberg, Melbourne, Australia More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.679AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Gastrin-releasing peptide (GRP) is a 10 amino acid amidated peptide which is widely distributed in the central nervous system and the gastrointestinal tract. It has now been shown to be mitogenic in a variety of cancers. The bioactive, amidated end-product GRPamide is processed from a 125 amino acid precursor, proGRP. While the Amidated GRP was originally thought to be the only biologically active product our laboratory has shown that bioactivity also resides in the proGRP C-terminal cleavage products. and may be the predominant form in cancer tissues. While there is preliminary evidence linking proGRP to prostate carcinogenesis, the nature of the receptors mediating any potential actions of proGRP has not been investigated, nor the type of peptides that are expressed or signalling pathways activated. METHODS 1. Receptor Binding Studies: Using radiolabelled amidated-GRP, proGRP47-68, and proGRP80-97, the receptor status of three prostate cancer cell lines were characterised by competitive binding assays. The cell lines used were hormone sensitive (LNcaP) and hormone refractory (PC-3, DU-145). 2. Sandwich Enzyme-linked Immunosorbent Assay (ELISA): three monoclonal antibodies recognising different domains (proGRP48-51, proGRP56-61 and proGRP84-88) were used to detect proGRP derived peptides in prostate cancer cell line extracts. 3. Reverse Phase High-performance Liquid Chromatography (HPLC): The proGRP immunoreactivity in the prostate cancer cell lines was characterised using HPLC. 4. Western Blot: PC-3 cells were treated with proGRP over various time points, and extracted. The extracts were used in western blots to see if MAP-kinase was activated RESULTS 1. Receptor Binding Studies: All three cell lines express receptors for proGRP but with far greater numbers on the androgen –independent PC-3 compared to the androgen-dependent LNCaP. 2. Sandwich Enzyme-linked Immunosorbent Assay (ELISA): Both PC-3 and LNCaP cells expressed proGRP with higher amounts detected in the hormone sensitive line. 3. Reverse Phase High-performance Liquid Chromatography (HPLC): HPLC confirmed the presence of C terminal reactive proGRP peptides. 4. Western Blot: Western blots of PC3 cells treated with proGRP showed activation of MAP Kinase pathway. CONCLUSIONS • Prostate cancer cell lines contain binding sites for both GRP and proGRP. • LNCaP and PC-3 cells express significant levels of proGRP peptides. • The nature and biological activity of the peptides that bind to these putative receptors require further investigation. • Treating cells with proGRP appears to activate MAP Kinase © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e456-e457 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chew-Lin Yip Heidelberg, Melbourne, Australia More articles by this author Rachele Lockie Heidelberg, Melbourne, Australia More articles by this author Joseph Ischia Heidelberg, Melbourne, Australia More articles by this author Liesl Ischia Heidelberg, Melbourne, Australia More articles by this author Oneel Patel Heidelberg, Melbourne, Australia More articles by this author Damien Bolton Melbourne, Australia More articles by this author Graham Baldwin Heidelberg, Melbourne, Australia More articles by this author Arthur Schulkes Heidelberg, Melbourne, Australia More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Analysis of an autoproteolytic activity of rice yellow mottle virus silencing suppressor P1

Ectopically expressed rice yellow mottle virus P1 fusion proteins were found to be cleaved in planta and in Escherichia coli. Cleavage takes place in the absence of bacterial protease activity, indicating that the P1 fusion is autocatalytically processed independently of host factors. N-terminal sequencing of the C-terminal cleavage product of transiently expressed P1/GFP (green fluorescence protein) in Nicotiana benthamiana showed that the cleavage site is located between the first two amino acids (aa) downstream of the P1 sequence. Mutagenesis experiments revealed that a phenylalanine to valine substitution at position 157 of the P1 aa sequence impairs proper cleavage, which is nearly unaffected by replacement of phenylalanine with tyrosine. Deletion of methionine(159) (first GFP aa residue) appeared to not affect P1/GFP cleavage. N-terminal P1-tagging with GFP turned out to impair autocleavage, whereas a small His-tag could not fully prevent cleavage. Additionally, a modified P1/GFP carrying an N-terminal deletion of 81 aa was not cleaved. These findings indicate that this region is involved in the proteolysis mechanism and that large N-terminal fusion partners might affect correct folding of the P1 necessary for self-catalysis.

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K(d) with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K(d) bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K(d) endocytosis. Upon increased expression of wild type APLP2, K(d) molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K(d) at the plasma membrane, facilitates uptake of K(d) in a clathrin-dependent manner, and routes the endocytosed K(d) to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knock-down experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.

Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

Ephrin-B2-induced Cleavage of EphB2 Receptor Is Mediated by Matrix Metalloproteinases to Trigger Cell Repulsion

EphB receptors provide crucial adhesive and repulsive signals during cell migration and axon guidance, but it is unclear how they switch between these opposing responses. Here we provide evidence of an important role for matrix metalloproteinases (MMPs) in repulsive EphB2 signaling. We found that EphB2 is cleaved by MMPs both in vitro and in vivo, and that this cleavage is induced by interaction with its ligand ephrin-B2. Our findings demonstrate that MMP-2/MMP-9-specific inhibition or cleavage-resistant mutations in the ectodomain of EphB2 can prevent EphB2-mediated cell-cell repulsion in HEK293 cells, and block ephrin-B1-induced growth cone withdrawal in cultured hippocampal neurons. Transient expression of wtEphB2, but not noncleavable EphB2-4/5 mutant, restored ephrin-B1-induced growth cone collapse and withdrawal in EphB-deficient neurons. The inhibition of EphB2 cleavage also had potent regulatory effects on EphB2 activity. This study provides the first evidence that MMP-mediated cleavage of EphB2 is induced by receptor-ligand interactions at the cell surface and that this event triggers cell-repulsive responses.

Porcine Dentin Sialophosphoprotein

Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects.

Direct analysis of tau from PSP brain identifies new phosphorylation sites and a major fragment of N‐terminally cleaved tau containing four microtubule‐binding repeats

Tangles containing hyperphosphorylated aggregates of insoluble tau are a pathological hallmark of progressive supranuclear palsy (PSP). Several phosphorylation sites on tau in PSP have been identified using phospho-specific antibodies, but no sites have been determined by direct sequencing due to the difficulty in enriching insoluble tau from PSP brain. We describe a new method to enrich insoluble PSP-tau and report eight phosphorylation sites [Ser46, Thr181, Ser202, Thr217, Thr231, Ser235, Ser396/Ser400 (one site) and Thr403/Ser404 (one site)] identified by mass spectrometry. We also describe a 35 kDa C-terminal tau fragment (tau35), lacking the N-terminus of tau but containing four microtubule-binding repeats (4R), that is present only in neurodegenerative disorders in which 4R tau is over-represented. Tau35 was readily detectable in PSP, corticobasal degeneration and 4R forms of fronto-temporal dementia with parkinsonism linked to chromosome 17, but was absent from control, Alzheimer's disease and Pick's disease brain. Our findings suggest the aggregatory characteristics of PSP-tau differ from those of insoluble tau in Alzheimer's disease brain and this might be related to the presence of a C-terminal cleavage product of tau.

Cellular localization of MAGI-1 caspase cleavage products and their role in apoptosis

MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein present at adherent and tight junctions, where it acts as a structural and signaling scaffold. During apoptosis, MAGI-1 is cleaved by caspases at Asp761 into N- and C-terminal cleavage products, allowing further dismantling of the cell junctions, one of the key features of apoptosis. Here, we investigated the cellular distribution and possible proapototic role of MAGI-1 caspase cleavage products. Full-length MAGI-1 exhibited submembrane localization, while the N-terminal caspase cleavage product of MAGI-1 is translocated to the cytosol and the C-terminal caspase cleavage product accumulates in the nucleus. When overexpressed in MDCK cells, both N- and C-terminal MAGI-1 caspase cleavage products exhibited minor proapoptotic activity, although their role in apoptosis is probably more passive.

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.

The Role of the N-terminal Domain of the Complement Fragment Receptor C5L2 in Ligand Binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.

The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment

Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl(-) channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.

Caspase-3 cleaves the formin-homology-domain-containing protein FHOD1 during apoptosis to generate a C-terminal fragment that is targeted to the nucleolus

The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD(616) site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation.