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Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr1065 on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr1065 in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr1065 (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr1065 phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr1065 phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation. [ABSTRACT FROM AUTHOR]

Phosphorylation of tropomyosin (Tm) has been shown to vary in mouse models of cardiac hypertrophy. Little is known about the in vivo role of Tm phosphorylation. This study examines the consequences of Tm dephosphorylation in the murine heart. Transgenic (TG) mice were generated with cardiac specific expression of α-Tm with serine 283, the phosphorylation site of Tm, mutated to alanine. Echocardiographic analysis and cardiomyocyte cross-sectional area measurements show that α-Tm S283A TG mice exhibit a hypertrophic phenotype at basal levels. Interestingly, there are no alterations in cardiac function, myofilament calcium (Ca2+) sensitivity, cooperativity, or response to β-adrenergic stimulus. Studies of Ca2+ handling proteins show significant increases in sarcoplasmic reticulum ATPase (SERCA2a) protein expression and an increase in phospholamban phosphorylation at serine 16, similar to hearts under exercise training. Compared with controls, the decrease in phosphorylation of α-Tm results in greater functional defects in TG animals stressed by transaortic constriction to induce pressure overload hypertrophy. This is the first study to investigate the in vivo role of Tmdephosphorylation under both normal and cardiac stress conditions, documenting a role for Tm dephosphorylation in the maintenance of acompensatedor physiological phenotype. Collectively, these results suggest that modification of theTm phosphorylation status in the heart, depending upon the cardiac state/condition, may modulate the development of cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting ∼60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5-9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1-3, interacts with Man8GlcNAc2 and Man9GlcNAc2 oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes. [ABSTRACT FROM AUTHOR]

Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP·Mg2+ binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp576 or Lys528 results in dramatically impaired binding affinities for ATP·Mg2+, a shift from ordered to random addition in co-substrate Binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp576 and Lys528 are important for ATP·Mg2+ binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp576 within the activating enzymes for other ubiquitin-like proteins. [ABSTRACT FROM AUTHOR]

Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-³H]ATP and [sup 125]I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [³H]adenylate and Uba3-[sup 125]I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with [sup 125]I-Nedd8 (k[sub cat] = 3.5 ± 0.2 s[sup -1] yielding K[sub m] values for ATP (103 ± 12 µM), [sup 125]I-Nedd8 (0.95 ' 0.18 µM), and HsUbc12 (43 ' 13 nM) similar to those for ubiquitin activation by Uba1. Wild type [sup 125]I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of [sup 125]I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a K[sub d] > 300 µM for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because [sup 125]IUbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields K[sub m] = 20 ± 9 µM and k[sub cat] = 0.9 ± 0.3 s[sup -1]. These observations demonstrate remarkable conservation in the mechanism of AppBp1Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme. [ABSTRACT FROM AUTHOR]

Describes the protein interactions within the n-ends ubiquitin ligation pathway. Employment of rate studies as a reporter function to probe protein-protein interactions; Presence of free ubiquitins in cells; Analysis of protein interactions within a ubiquitin ligation pathway.

Previously we reported that adipocyte SNAP23 (synaptosome- associated protein of 23 kDa) deficiency blocks the activation of macroautophagy, leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo. Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion, BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, whereas in the nutrient-depleted state, BAX binds to SNAP23 to form a ternary BAX-SNAP23-ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program. [ABSTRACT FROM AUTHOR]

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2C~4A mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2, Proteasomal inhibitors rescued both MDM2 and H2O2-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis. [ABSTRACT FROM AUTHOR]

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells. [ABSTRACT FROM AUTHOR]

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes. [ABSTRACT FROM AUTHOR]

We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients. [ABSTRACT FROM AUTHOR]

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosisspecific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle. [ABSTRACT FROM AUTHOR]

Inhibition of apoptosis is critical for carcinogenesis. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both intrinsic and extrinsic apoptosis pathways. Although normally expressed in striated myocytes and neurons, ARC is markedly induced in a variety of primary human epithelial cancers and renders cancer cells resistant to killing. The mechanisms that mediate the induction of ARC in cancer are unknown. Herein we demonstrate that increases in ARC abundance are stimulated by Ras through effects on transcription and protein stability. Overexpression of activated N-Ras or H-Ras in normal cells is sufficient to increase ARC mRNA and protein levels. Similarly, transgenic expression of activated H-Ras induces ARC in both the normal mammary epithelium and resulting tumors of intact mice. Conversely, knockdown of endogenous N-Ras in breast and colon cancer cells significantly reduces ARC mRNA and protein levels. The promoter of the Nol3 locus, encoding ARC, is activated by N-Ras and H-Ras in a MEK/ERK-dependent manner. Ras also stabilizes ARC protein by suppressing its polyubiquitination and subsequent proteasomal degradation. In addition to the effects of Ras on ARC abundance, ARC mediates Ras-induced cell survival and cell cycle progression. Thus, Ras induces ARC in epithelial cancers, and ARC plays a role in the oncogenic actions of Ras. [ABSTRACT FROM AUTHOR]

The C terminus of the β2-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the β2-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62 Å, respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the β2-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling. [ABSTRACT FROM AUTHOR]

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death. [ABSTRACT FROM AUTHOR]

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured. [ABSTRACT FROM AUTHOR]

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (α2β2) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. L., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587–5598). An annular gap of ∼90 Å separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of ∼75 Å between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations orE3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. [ABSTRACT FROM AUTHOR]

Presents a study indicating that the sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes. Description of a novel effect of ceramide, namely the induction of transbilayer lipid movements; Novel assays developed to detect transbilayer lipid movement; Role of sphingomyelinase activity in inducing ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles.

Reports on the combination of fluorescence microscopy and fluorescence resonance energy transfer (FRET) techniques to define the relative positions of the transcriptive activation (TA) and bZIP transcription factor domains in dimers of C/EBPα at different subregions of living cell nuclei.