CP12 from Chlamydomonas reinhardtii, a Permanent Specific ��Chaperone-like�� Protein of Glyceraldehyde-3-phosphate Dehydrogenase

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A₄ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific “chaperone-like protein” for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A₂B₂ GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria (1, 2), higher plants (3), the diatom Asterionella formosa (4, 5), and green (1) and red algae (6). It allows the formation of a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),³ two key enzymes of the Calvin cycle pathway, and was recently shown to interact with fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway (7). The phosphoribulokinase·GAPDH·CP12 complex has been extensively studied in Chlamydomonas reinhardtii (8, 9) and in Arabidopsis thaliana (10, 11). In the green alga C. reinhardtii, the interaction between CP12 and GAPDH is strong (8). GAPDH may exist as a homotetramer composed of four GapA subunits (A₄) in higher plants, cyanobacteria, and green and red algae (6, 12), but in higher plants, it can also exist as a heterotetramer (A₂B₂), composed of two subunits, GapA and GapB (13, 14). GapB, up to now, has exclusively been found in Streptophyta, but recently two prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, were also shown to possess a GapB gene, whereas CP12 is missing (15). The GapB subunit is similar to the GapA subunit but has a C-terminal extension containing two redox-regulated cysteine residues (16). Thus, although the A₄ GAPDHs lack these regulatory cysteine residues (13, 14, 17–20), they are also redox-regulated through its interaction with CP12, since the C terminus of this small protein resembles the C-terminal extension of the GapB subunit. The regulatory cysteine residues for GapA are thus supplied by CP12, as is well documented in the literature (1, 8, 11, 16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs) (21–26). The amino acid composition of these proteins causes them to have no or few secondary structures. Their total or partial lack of structure and their high flexibility allow them to be molecular adaptors (27, 28). They are often able to bind to several partners and are involved in most cellular functions (29, 30). Recently, some IUPs have been described in photosynthetic organisms (31, 32).There are many functional categories of IUPs (22, 33). They can be, for instance, involved in permanent binding and have (i) a scavenger role, neutralizing or storing small ligands; (ii) an assembler role by forming complexes; and (iii) an effector role by modulating the activity of a partner molecule (33). These functions are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating its redox properties (8, 34, 35), and can also bind a metal ion (36, 37). IUPs can also bind transiently to partners, and some of them have been found to possess a chaperone activity (31, 38). This chaperone function was first shown for α-synuclein (39) and for α-casein (40), which are fully disordered. The amino acid composition of IUPs is less hydrophobic than those of soluble proteins; hence, they lack hydrophobic cores and do not become insoluble when heated. Since CP12 belongs to this family, we tested if it was resistant to heat treatment and finally, since it is tightly bound to GAPDH, if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate (41–44) and consequently a substrate commonly used in chaperone studies (45, 46).Unlike chaperones, which form transient, dynamic complexes with their protein substrates through hydrophobic interactions (47, 48), CP12 forms a stable complex with GAPDH. The interaction involves the C-terminal part of the protein and the presence of negatively charged residues on CP12 (35). However, only a site-directed mutagenesis has been performed to characterize the interaction site on GAPDH. Although the mutation could have an indirect effect, the residue Arg-197 was shown to be a good candidate for the interaction site (49).In this report, we accordingly used proteolysis experiments coupled with mass spectrometry to detect which regions of GAPDH are protected by its association with CP12. To conclude, the aim of this report was to characterize a chaperone function of CP12 that had never been described before and to map the interaction site on GAPDH using an approach that does not involve site-directed mutagenesis.     

WISP1, a Pro-mitogenic, Pro-survival Factor, Mediates Tumor Necrosis Factor-�� (TNF-��)-stimulated Cardiac Fibroblast Proliferation but Inhibits TNF-��-induced Cardiomyocyte Death

WNT1-inducible signaling pathway protein-1 (WISP1), a member of the CYR61/CTGF/Nov family of growth factors, can mediate cell growth, transformation, and survival. Previously we demonstrated that WISP1 is up-regulated in post-infarct heart, stimulates cardiac fibroblast proliferation, and is induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Here we investigated (i) the localization of TNF-α and WISP1 in post-infarct heart, (ii) the mechanism of TNF-α-mediated WISP1 induction in primary human cardiac fibroblasts (CF), (iii) the role of WISP1 in TNF-α-mediated CF proliferation and collagen production, and (iv) the effects of WISP1 on TNF-α-mediated cardiomyocyte death. TNF-α and WISP1 expressions were increased in the border zones and non-ischemic remote regions of the post-ischemic heart. In CF, TNF-α potently induced WISP1 expression in cyclic AMP response element-binding protein (CREB)-dependent manner. TNF-α induced CREB phosphorylation in vitro and DNA binding and reporter gene activities in vivo. TNF-α induced CREB activation via ERK1/2, and inhibition of ERK1/2 and CREB blunted TNF-α-mediated WISP1 induction. Most importantly, WISP1 knockdown attenuated TNF-α stimulated collagen production and CF proliferation. Furthermore, WISP1 attenuated TNF-α-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and pro-survival effects for WISP1 in myocardial constituent cells. Our results suggest that a TNF-α/WISP1 signaling pathway may contribute to post-infarct cardiac remodeling, a condition characterized by fibrosis and progressive cardiomyocyte loss.Acute myocardial infarction (MI)² is a major cause of morbidity and mortality worldwide. After acute MI, the two principal determinants of patient outcome are myocardial infarct size and the extent of left ventricular (LV) remodeling (1–3). Infarct size is determined during the acute phase immediately post-MI. LV remodeling on the other hand is a continuous and maladaptive process characterized by progressive left ventricular hypertrophy, fibrosis, ventricular dilatation, and by the gradual deterioration of cardiac performance, leading ultimately to congestive heart failure (1–3).Numerically, fibroblasts are the major cell type in the heart, but they constitute a smaller total volume compared with cardiomyocytes (4). Fibroblasts are associated primarily with modulation of extracellular matrix and tissue healing/repair (4–6). Fibroblasts secrete collagens, fibrillins, fibronectin, laminin, and matrix metalloproteinases and, thus, are responsible for the maintenance of connective tissue homeostasis (4–7). We and others have shown that primary cardiac fibroblasts also produce various cytokines, chemokines, and growth factors (4–6, 8, 9), which tightly regulate the physiological function of the cells. Under pathological conditions, however, where the expression of cytokines and growth factors is significantly altered, the fibroblasts can undergo differentiation, migration, and proliferation, resulting in pathological fibrosis because of excessive accumulation of collagens and other ECM proteins (4–6).The six members of the CCN (CYR61/CTGF/Nov) family of growth factors (Cyr61 (cysteine-rich 61, CCN1), CTGF (connective tissue growth factor, CCN2), Nov (nephroblastoma-overexpressed, CCN3), WISP1 (WNT1-inducible signaling pathway protein-1, CCN4), WISP2 (CCN5), and WISP3 (CCN6)) are involved in a number of cellular processes, including adhesion, migration, and proliferation (10–12). Although their roles in angiogenesis and oncogenesis are well characterized, with the exception of CTGF, their precise contribution to myocardial remodeling is unknown. Recently we demonstrated that whereas WISP1 is expressed in the heart at low basal levels, permanent occlusion of the left anterior descending coronary artery significantly up-regulates its expression in the non-ischemic myocardium (13). Furthermore, we have found that WISP1 exerts both pro-hypertrophic and pro-mitogenic effects in vitro, stimulating Akt-dependent cardiomyocyte growth, as well as collagen synthesis and fibroblast proliferation (13). Because cardiomyocyte hypertrophy and fibroblast proliferation play central roles in remodeling and WISP1 regulates these two critical processes, it is plausible that WISP1 also mediates cardiac remodeling after myocardial ischemia, infarction, and inflammation. However, the mechanisms involved in the induction and regulation of WISP1 under these conditions have not been well characterized.The proinflammatory cytokine tumor necrosis factor (TNF)-α is expressed at low basal levels in the normal myocardium but is significantly up-regulated after infection, inflammation, and injury (14, 15). Although low levels are considered cytoprotective (16, 17), aberrant expression of TNF-α during myocardial ischemic injury and inflammation induces cardiomyocyte death, hypertrophy of surviving cardiomyocytes, contractile dysfunction, fibroblast proliferation, fibrosis, and adverse remodeling (14–17). TNF-α exerts its biological effects via two cell surface receptors, TNFR1 (55 kDa) and TNFR2 (75 kDa) (18). TNFR1, which is more abundantly expressed in the heart, appears to be the main signaling receptor for TNF-α and is implicated in transmitting its deleterious effects (18). On the other hand, signaling through TNFR2 appears to exert a protective effect in the heart (18, 19). Of note, all myocardial constituent cells, including fibroblasts, express both TNFR1 and TNFR2 and, therefore, are targets of TNF-α.We have recently demonstrated that TNF-α induces WISP1 expression in cardiomyocytes (13). However, neither its localization in post-infarct myocardium, its role in TNF-α-mediated cardiac fibroblast (CF) proliferation and collagen production, nor its effects on TNF-α-mediated cardiomyocyte death are known. Our results show that both TNF-α and WISP1 are localized in the border zone and the non-infarct remote region in post-infarct myocardium. Furthermore, TNF-α induces WISP1 expression in CF via a TNFR2/MEK1/ERK1/2/CREB pathway and stimulates fibroblast collagen production in part via WISP1. In addition, WISP1 exerts pro-survival effects in cardiomyocytes, antagonizing TNF-α-mediated cardiomyocyte death. Collectively, these results indicate that WISP1 exerts pro-mitogenic and pro-survival effects in cardiac cell type-specific manner, and TNF-α/WISP1 signaling may be an important contributing mechanism in post-infarct cardiac remodeling.     

Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

ULK1��ATG13��FIP200 Complex Mediates mTOR Signaling and Is Essential for Autophagy

Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvation-induced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1·ATG13·FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process whereby long-lived proteins and damaged organelles are shuttled to lysosomes for degradation. This process is conserved in all eukaryotes. Under normal growth conditions a housekeeping level of autophagy exists. Under stress, such as nutrient starvation, autophagy is strongly induced resulting in the engulfment of cytosolic components and organelles in specialized double-membrane structures termed autophagosomes. Following fusion of the outer autophagosomal membrane with lysosomes, the inner membrane and its cytoplasmic cargo are degraded and recycled (1–3). Recent work has implicated autophagy in many disease pathologies, including cancer, neurodegeneration, as well as in eliminating intracellular pathogens (4–8).The morphology of autophagy was first described in mammalian cells over 50 years ago (9). However, it is only recently through yeast genetic screens, that multiple autophagy-related (ATG) genes have been identified (10–12). The yeast ATG proteins have been classified into four major groups: the Atg1 protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex (13). Even though many ATG genes are now known, most of which have functional homologs in mammalian cells (14, 15), the molecular mechanism by which they sense the initial triggers and subsequently dictate autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein kinase and a key autophagy-regulator (16). Atg1 is complexed to at least two other proteins during autophagy, Atg13 and Atg17, both of which are required for normal Atg1 function and autophagosome generation (17–19). Classical signaling pathways such as the cAMP-dependent kinase (PKA) pathway or the Tor kinase pathway appear to converge upon this complex, placing Atg1 at an early stage during autophagosome biogenesis (20–22). Atg1 phosphorylation by PKA blocks its association with the forming autophagosome (21), while the Tor pathway hyperphosphorylates Atg13 causing a reduced affinity of Atg13 for Atg1, resulting in repression of autophagy (17, 19). In contrast, nutrient starvation or inhibition of Tor leads to dephosphorylation of Atg13 thus increased Atg1 complex formation and kinase activity, resulting in stimulation of autophagy (19). Surprisingly, the physiological substrates of Atg1 kinase have not been identified; thus how Atg1 transduces upstream autophagic signaling is undefined. Recently, mammalian homologs of Atg1 have been identified as ULK1 and ULK2 (Unc-51-like kinase)² (23–25). ULK1 and ULK2 are ubiquitously expressed and localize to the isolation membrane, or forming autophagosome, upon nutrient starvation (25); RNAi-mediated depletion of ULK1 in HEK293 cells compromises autophagy (23, 24). The exact role of ULK1 versus ULK2 in autophagy is unclear, and it is possible some redundancy exists between the two isoforms (26).Given the conservation of autophagy from yeast to man, it is interesting to note that no mammalian counterpart to yeast Atg13 or Atg17 had been identified until very recently. The protein FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) was identified as an autophagy-essential binding partner of both ULK1 and ULK2 (25), and it has been speculated that FIP200 might be the equivalent of yeast Atg17, despite low sequence similarity (25, 27).In this study, we delve deeper into the molecular regulation of ULK1 to gain a better insight into how mammalian signaling pathways affect autophagy initiation. We describe here the identification of a triple complex consisting of ULK1, FIP200, and the mammalian equivalent of Atg13. This complex is required not only for localization of ULK1 to the isolation membrane but also for maximal kinase activity. In addition, both ATG13 and ULK1 are kinase substrates in the mTOR pathway and thus might function to sense nutrient starvation. Therefore, this study defines the role of mammalian ULK1-ATG13-FIP200 complex in mediating the initial autophagic triggers and to transduce the signal to the core autophagic machinery.     

Nitro-fatty Acid Metabolome: Saturation, Desaturation, ��-Oxidation, and Protein Adduction

Nitrated derivatives of fatty acids (NO₂-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO₂-FA signal transduction lacks insight into how or if NO₂-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO₂) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO₂ or metabolites were detected under basal conditions, whereas administered 18:1-NO₂ is rapidly adducted to plasma thiol-containing proteins and glutathione. NO₂-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO₂ as metabolites. Additionally, a significant proportion of 18:1-NO₂ and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO₂ hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO₂ detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO₂ release from glutathione-18:1-NO₂ adducts, supports that reversible and exchangeable NO₂-FA-thiol adducts occur under biological conditions. After administration of [³H]18:1-NO₂, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration.The reaction of unsaturated fatty acids with nitric oxide (•NO)- and nitrite species, including nitrogen dioxide (•NO₂), peroxynitrite (ONOO^–), and nitrous acid (HNO₂), yields a complex array of oxidized and nitrated products (1–4). The mechanisms of biological fatty acid nitration, the structural isomer distribution of nitrated fatty acids (NO₂-FAs)² and the signaling actions of specific NO₂-FA regioisomers remain incompletely characterized. Current data reveal that, during fatty acid oxidation and nitration, vinyl nitro regioisomers represent a component of these products that display distinctive chemical reactivities and receptor-dependent signaling actions. Here, we investigate the metabolic fate of the nitroalkene derivative of oleic acid (1, 2).Unsaturated fatty acid nitration was first described in model studies of air-pollutant-induced lipid oxidation where lipids were exposed to high concentrations of •NO₂ (5, 6). More recently nitrated unsaturated fatty acids have been reported as products of acidic reactions of , radical chain termination reactions induced by •NO (7–10), and the oxidation of to •NO₂ by the leukocyte-derived enzyme myeloperoxidase (1). Various mechanisms can mediate the formation of nitroalkene derivatives of unsaturated fatty acids (11), including homolytic attack of •NO₂ (12), reaction of •NO₂ with a pre-existing fatty acid carbon-centered radical (2, 13), and the protonation of nitrite under acidic conditions (pH 5.5 and lower) to yield an array of HNO₂-derived nitrating species (3, 14). The conditions promoting fatty acid nitration by •NO and species (low oxygen tension, radical formation, and low pH) are not expected to be broadly distributed systemically (e.g. in plasma or extracellular fluids). Rather, nitration reactions will preferably occur during inflammatory or metabolic stress in microenvironments such as the intermembrane space of mitochondria, the low pH environment of the digestive tract, and activated macrophage and neutrophil-rich compartments. Moreover, the acidic, replete and low O₂ tension conditions that promote nitration reactions are characteristic of inflammatory loci. Although multiple reactions leading to accelerated formation of nitrating species occur at specific anatomic sites, plasma levels of nitrated fatty acids are expected to be low due to events described herein.Robust electrophilic reactivity and avid nuclear lipid receptor ligand activity have conferred to the class of fatty acid nitroalkene derivatives potent anti-inflammatory properties that occur predominantly via non-cGMP-dependent mechanisms. Nitro derivatives of oleic and linoleic acid inhibit leukocyte and platelet activation (15), vascular smooth muscle proliferation (16), lipopolysaccharide-stimulated macrophage cytokine secretion (17), activate peroxisome proliferator-activated receptor-γ (1, 18), and induce endothelial heme oxygenase 1 expression (19). NO₂-FA also potently modulate nuclear factor-erythroid 2-related factor 2/Kelch-like ECH-associating protein 1 (Nrf2/Keap1) (16, 17) and nuclear factor κB (NFκB)-regulated inflammatory signaling (17). Previous observations of the •NO-mediated, cGMP-dependent vessel relaxation induced by NO₂-FA were made under serum- and lipid-free conditions. More recently, it has been appreciated that micellar and membrane stabilization of NO₂-FA prevents Nef-like aqueous decay reactions and consequent •NO release, supporting that the predominant signaling actions mediated by NO₂-FA are •NO and cGMP-independent (20, 21).Current data indicate that electrophilic adduction of biological targets primarily accounts for NO₂-FA signal transduction. The high electronegativity of NO₂ substituents, when bound to an alkenyl carbon of fatty acids, confers an electrophilic nature to the adjacent β-carbon and enables Michael addition reaction with nucleophiles such as protein His and Cys residues. This process, termed nitroalkylation (22), results in the clinically detectable and reversible adduction of the nucleophilic thiol of glutathione (GSH) and both cysteine and histidine residues of glyceraldehyde-3-phosphate dehydrogenase (23). Furthermore, inhibition of NFκB signaling occurs via nitroalkylation of p65 subunit thiols (17), and recent findings reveal that NO₂-FA activation of peroxisome proliferator-activated receptor-γ is uniquely induced by covalent nitroalkylation of the ligand binding domain Cys-285.³Multiple reports support the endogenous generation and presence of nitrated fatty acids (1, 24), first observed in bovine papillary muscles as a vicinal nitrohydroxyeicosatetraenoic acid (25). Nitrolinoleate has been detected in human blood plasma and cholesteryl nitrolinoleate in human plasma and lipoproteins (4, 26), with hyperlipidemic and post-prandial conditions elevating plasma levels of NO₂-FA. Further support for the inflammatory generation of NO₂-FA comes from lipopolysaccharide and interferon-γ-activated murine J774.1 macrophages, where increased nitration of the acyl chain of cholesteryl linoleate was paralleled by increased macrophage expression and activity of nitric-oxide synthase 2 (27).To date, insight into the mechanisms of nitroalkene signaling actions overshadows knowledge of the generation, trafficking, and metabolism of nitroalkenes in vivo. Appreciating that NO₂-FA derivatives are detectable clinically, and that their levels increase following •NO-dependent oxidative reactions (4, 28), challenges still exist in their routine detection. Because the in vivo administration of NO₂-FA may exert anti-inflammatory benefit, the disposition and metabolite profiles of these species in vivo is of relevance. Here we report that only 2.4% of nitrooctadecenoic acid (18:1-NO₂) is immediately detectable in the vascular compartment as native 18:1-NO₂ upon intravenous injection in mice, with the remaining pool of 18:1-NO₂ (a) reversibly bound to plasma and tissue thiols via Michael addition; (b) metabolized to nitro-octadecanoic acid (18:0-NO₂) and nitro-octadecadienoic acid (18:2-NO₂); and (c) catabolized by hepatic β-oxidation following thioester formation with coenzyme A.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.     

Insulin Resistance in Striated Muscle-specific Integrin Receptor ��1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1-5). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6, 7).Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS)² phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8-11). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12, 13). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14-18). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20, 21). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22-24).In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25, 26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27-33). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle.