Journal of Biological Chemistry current issue

[Classics] How Aspirin Interferes with Cyclooxygenase Activity: the Work of William L. Smith

Kresge, N., Simoni, R. D., Hill, R. L. — 2008-05-09

[Minireviews] Molecular Machinery of Mitochondrial Fusion and Fission

Westermann, B. — 2008-05-09

[Accelerated Publications] Requirement of Inositol 1,4,5-Trisphosphate Receptors for Tumor-mediated Lymphocyte Apoptosis

Steinmann, C., Landsverk, M. L., Barral, J. M., Boehning, D. — 2008-05-09

Tumor cells strategically down-regulate Fas receptor expression to evade immune attack and up-regulate expression of Fas ligand to promote apoptosis of infiltrating T lymphocytes. Many pathways leading to apoptotic cell death require calcium release from inositol 1,4,5-trisphosphate receptors (IP₃Rs). Here, we show that Fas-dependent killing of Jurkat T lymphoma cells by SW620 colon cancer cells requires calcium release from IP₃R. General suppression of IP₃R signaling significantly reduced SW620-mediated Jurkat cell apoptosis. Significantly, a specific inhibitor of apoptotic calcium release from IP₃R strongly blocked lymphocyte apoptosis. Thus, selective pharmacological targeting of apoptotic calcium release from IP₃R may enhance tumor cell immunogenicity.

[Lipids and Lipoproteins: Metabolism, Regulation, and Signaling] The Molecular Basis of Retinoid Absorption: A Genetic Dissection

2008-05-09

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.

[Membrane Transport, Structure, Function, and Biogenesis] Arabidopsis ANTR1 Is a Thylakoid Na+-dependent Phosphate Transporter: FUNCTIONAL CHARACTERIZATION IN ESCHERICHIA COLI

Pavon, L. R., Lundh, F., Lundin, B., Mishra, A., Persson, B. L., Spetea, C. — 2008-05-09

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na⁺-dependent inorganic phosphate (P_i) transporters. The function of ANTR1 as a Na⁺-dependent P_i transporter was demonstrated by heterologous expression and uptake of radioactive P_i into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated P_i uptake in a pH- and Na⁺-dependent manner as compared with the control cells. Among various tested effectors, P_i was the preferred substrate. Although it competed with the uptake of P_i, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a P_i transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of P_i produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H⁺ electrochemical gradient storage.

[Protein Structure and Folding] A Limited Role for Disulfide Cross-linking in the Aggregation of Mutant SOD1 Linked to Familial Amyotrophic Lateral Sclerosis

Karch, C. M., Borchelt, D. R. — 2008-05-09

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.

[Lipids and Lipoproteins: Metabolism, Regulation, and Signaling] Protection from High Fat Diet-induced Increase in Ceramide in Mice Lacking Plasminogen Activator Inhibitor 1

Shah, C., Yang, G., Lee, I., Bielawski, J., Hannun, Y. A., Samad, F. — 2008-05-09

Obesity increases the risk for metabolic and cardiovascular disease, and adipose tissue plays a central role in this process. Ceramide, the key intermediate of sphingolipid metabolism, also contributes to obesity-related disorders. We show that a high fat diet increased ceramide levels in the adipose tissues and plasma in C57BL/6J mice via a mechanism that involves an increase in gene expression of enzymes mediating ceramide generation through the de novo pathway (e.g. serine palmitoyltransferase) and via the hydrolysis of sphingomyelin (acid sphingomyelinase and neutral sphingomyelinase). Although the induction of total ceramide in response to the high fat diet was modest, dramatic increases were observed for C16, C18, and C18:1 ceramides. Next, we investigated the relationship of ceramide to plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of plasminogen activation and another key player in obesity. PAI-1 is consistently elevated in obesity and thought to contribute to increased artherothrombotic events and more recently to obesity-mediated insulin resistance. Interestingly, the changes in ceramide were attenuated in mice lacking PAI-1. Mechanistically, mice lacking PAI-1 were protected from diet-induced increase in serine palmitoyltransferase, acid sphingomyelinase, and neutral sphingomyelinase mRNA, providing a mechanistic link for decreased ceramide in PAI-1^–/– mice. The decreases in plasma free fatty acids and adipose tumor necrosis factor- in PAI-1^–/– mice may have additionally contributed indirectly to improvements in ceramide profile in these mice. This study has identified a novel link between sphingolipid metabolism and PAI-1 and also suggests that ceramide may be an intermediary molecule linking elevated PAI-1 to insulin resistance.

[Dna: Replication, Repair, Recombination, and Chromosome Dynamics] RNF8-dependent and RNF8-independent Regulation of 53BP1 in Response to DNA Damage

Sakasai, R., Tibbetts, R. — 2008-05-09

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G₂/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.

[Membrane Transport, Structure, Function, and Biogenesis] Phage T5 Straight Tail Fiber Is a Multifunctional Protein Acting as a Tape Measure and Carrying Fusogenic and Muralytic Activities

2008-05-09

We report a bioinformatic and functional characterization of Pb2, a 121-kDa multimeric protein that forms phage T5 straight fiber and is implicated in DNA transfer into the host. Pb2 was predicted to consist of three domains. Region I (residues 1–1030) was mainly organized in coiled coil and shared features of tape measure proteins. Region II (residues 1030–1076) contained two -helical transmembrane segments. Region III (residues 1135–1148) included a metallopeptidase motif. A truncated version of Pb2 (Pb2-Cterm, residues 964–1148) was expressed and purified. Pb2-Cterm shared common features with fusogenic membrane polypeptides. It formed oligomeric structures and inserted into liposomes triggering their fusion. Pb2-Cterm caused β-galactosidase release from Escherichia coli cells and in vitro peptidoglycan hydrolysis. Based on these multifunctional properties, we propose that binding of phage T5 to its receptor triggers large conformational changes in Pb2. The coiled coil region would serve as a sensor for triggering the opening of the head-tail connector. The C-terminal region would gain access to the host envelope, permitting the local degradation of the peptidoglycan and the formation of the DNA pore by fusion of the two membranes.

[Mechanisms Of Signal Transduction] Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury

Hanawa, N., Shinohara, M., Saberi, B., Gaarde, W. A., Han, D., Kaplowitz, N. — 2008-05-09

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165–178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H₂O₂ release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H₂O₂ or inhibitors (e.g. antimycin) that cause increased H₂O₂ release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.

[Protein Synthesis, Post-Translational Modification, and Degradation] Opposite Regulation of CD36 Ubiquitination by Fatty Acids and Insulin: EFFECTS ON FATTY ACID UPTAKE

Smith, J., Su, X., El-Maghrabi, R., Stahl, P. D., Abumrad, N. A. — 2008-05-09

FAT/CD36 is a membrane scavenger receptor that facilitates long chain fatty acid uptake by muscle. Acute increases in membrane CD36 and fatty acid uptake have been reported in response to insulin and contraction. In this study we have explored protein ubiquitination as one potential mechanism for the regulation of CD36 level. CD36 expressed in Chinese hamster ovary (CHO) or HEK 293 cells was found to be polyubiquitinated via a process involving both lysines 48 and 63 of ubiquitin. Using CHO cells expressing the insulin receptor (CHO/hIR) and CD36, it is shown that addition of insulin (100 nm, 10 and 30 min) significantly reduced CD36 ubiquitination. In contrast, ubiquitination was strongly enhanced by fatty acids (200 µm palmitate or oleate, 2 h). Similarly, endogenous CD36 in C2C12 myotubes was ubiquitinated, and this was enhanced by oleic acid treatment, which also reduced total CD36 protein in cell lysates. Insulin reduced CD36 ubiquitination, increased CD36 protein, and inhibited the opposite effects of fatty acids on both parameters. These changes were paralleled by changes in fatty acid uptake, which could be blocked by the CD36 inhibitor sulfosuccinimidyl oleate. Mutation of the two lysine residues in the carboxyl-terminal tail of CD36 markedly attenuated ubiquitination of the protein expressed in CHO cells and was associated with increased CD36 level and enhanced oleate uptake and incorporation into triglycerides. In conclusion, fatty acids and insulin induce opposite alterations in CD36 ubiquitination, modulating CD36 level and fatty acid uptake. Altered CD36 turnover may contribute to abnormal fatty acid uptake in the insulin-resistant muscle.

[Transcription, Chromatin, and Epigenetics] The Proinflammatory Cytokine, Interleukin-6, Up-regulates Calcium-sensing Receptor Gene Transcription via Stat1/3 and Sp1/3

Canaff, L., Zhou, X., Hendy, G. N. — 2008-05-09

Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5'-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated ~2–3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis.

[Transcription, Chromatin, and Epigenetics] Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Nabavi, S., Nazar, R. N. — 2008-05-09

Although the termination of transcription and 3' RNA processing of the eukaryotic mRNA has been linked to a polyadenylation signal and a transcript cleavage process, much less is known about the termination or processing of nonpolyadenylated RNA polymerase II transcripts. An efficiently expressed plasmid-based expression system was used to study the termination and processing of Schizosaccharomyces pombe U3 small nucleolar RNA (snoRNA) transcripts in vivo. The termination assay was linked to cell transformation, and restriction fragment length polymorphism was used to determine levels of plasmid-derived U3 snoRNA. Mutation analyses in vivo indicate that the maturation of the 3' end is not directly dependent on an external cis-acting sequence or structure; rather, it is dependent on a transcript cleavage that can occur hundreds or even thousands of nucleotides downstream of the mature U3 snoRNA sequence. Similarly, termination is dependent on the same transcript cleavage that is localized in a hairpin structure that normally follows the 3' end of the U3 snoRNA but that also can be moved hundreds or thousands of nucleotides downstream. Both processes, however, can be induced simultaneously and equally efficiently with a single unrelated Pac1 endonuclease-labile structure. The results support a "reversed torpedoes" model in which a single cleavage allows exonucleases and/or other protein factors access to the transcript leading to transcription termination in one direction and RNA maturation in the other direction.

[Transcription, Chromatin, and Epigenetics] Histone Code Modifications Repress Glucose Transporter 4 Expression in the Intrauterine Growth-restricted Offspring

Raychaudhuri, N., Raychaudhuri, S., Thamotharan, M., Devaskar, S. U. — 2008-05-09

We examined transcriptional and epigenetic mechanism(s) behind diminished skeletal muscle GLUT4 mRNA in intrauterine growth-restricted (IUGR) female rat offspring. An increase in MEF2D (inhibitor) with a decline in MEF2A (activator) and MyoD (co-activator) binding to the glut4 promoter in IUGR versus control was observed. The functional role of MEF2/MyoD-binding sites and neighboring three CpG clusters in glut4 gene transcription was confirmed in C2C12 muscle cells. No differential methylation of these three and other CpG clusters in the glut4 promoter occurred. DNA methyltransferase 1 (DNMT1) in postnatal, DNMT3a, and DNMT3b in adult was differentially recruited with increased MeCP2 (methyl CpG-binding protein) concentrations to bind the IUGR glut4 gene. Covalent modifications of the histone (H) code consisted of H3.K14 de-acetylation by recruitment of histone deacetylase (HDAC) 1 and enhanced association of HDAC4 enzymes. This set the stage for Suv39H1 methylase-mediated di-methylation of H3.K9 and increased recruitment of heterochromatin protein 1, which partially inactivates postnatal and adult IUGR glut4 gene transcription. Further increased interactions in the adult IUGR between DNMT3a/DNMT3b and HDAC1 and MEF2D and HDAC1/HDAC4 and decreased association between MyoD and MEF2A existed. We conclude that epigenetic mechanisms consisting of histone code modifications repress skeletal muscle glut4 transcription in the postnatal period and persist in the adult female IUGR offspring.

[Protein Structure and Folding] Scrambled Isomers as Key Intermediates in the Oxidative Folding of Ligand Binding Module 5 of the Low Density Lipoprotein Receptor

Arias-Moreno, X., Arolas, J. L., Aviles, F. X., Sancho, J., Ventura, S. — 2008-05-09

The ligand binding module five (LA5) of the low density lipoprotein receptor is a small, single-domain protein of 40 residues and three disulfide bonds with a calcium binding motif that is essential for its structure and function. Several mutations in LA5 have been reported to cause familial hypercholesterolemia by impairing a proper folding of the module. The current study reports the oxidative folding and reductive unfolding pathways of wild type and mutant LA5 modules through kinetic and structural analysis of the trapped intermediates. Wild type LA5 folding involves an initial phase of nonspecific packing where the sequential oxidation of its cysteines gives rise to complex equilibrated populations of intermediates. In the presence of calcium, the attainment of a coordination-competent conformation becomes the rate-limiting step of folding while binding of the ion funnels both thermodynamically and kinetically the folding reaction toward the native state. In the absence of calcium, a scrambled isomer (termed Xa) constitutes the global free energy minimum of the folding process. Xa and the native form are stable, inter-convertible species whose relative populations at equilibrium appear displaced in disease-linked mutants toward the scrambled form. Because stable scrambled isomers such as Xa avoid the exposition of reactive cysteines in misfolded modules, they might constitute a strategy to prevent wrong interactions with other domains during folding of the receptor. Comparison of the folding pathways of wild type and mutant LA5 provides the molecular basis to understand how LA modules fold into a functional conformation or upon mutation misfold and lead to disease.

[Molecular Basis Of Cell and Developmental Biology] Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

Stahl, M., Uemura, K., Ge, C., Shi, S., Tashima, Y., Stanley, P. — 2008-05-09

Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1^–/– ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1^–/– cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.

[Glycobiology and Extracellular Matrices] Interaction of Pro-matrix Metalloproteinase-9/Proteoglycan Heteromer with Gelatin and Collagen

Malla, N., Berg, E., Uhlin-Hansen, L., Winberg, J.-O. — 2008-05-09

Previously we have shown that THP-1 cells synthesize matrix metalloproteinase-9 (MMP-9) where a fraction of the enzyme is strongly linked to a proteoglycan (PG) core protein. In the present work we show that these pro-MMP-9·PG heteromers have different biochemical properties compared with the monomeric form of pro-MMP-9. In these heteromers, the fibronectin II-like domain in the catalytic site of the enzyme is hidden, and the fibronectin II-like-mediated binding to gelatin and collagen is prevented. However, a fraction of the pro-MMP-9·PG heteromers interacted with gelatin and collagen. This interaction was not through the chondroitin sulfate (CS) part of the PG molecule but, rather, through a region in the PG core protein, a new site induced by the interaction of pro-MMP-9 and the PG core protein, or a non-CS glycosaminoglycan part of the PG molecule. The interaction between pro-MMP-9·PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9·PG heteromers and pro-MMP-9 with approximately the same affinity. Removal of CS chains from the PG part of the heteromers did not affect the binding to gelatin and collagen. Although the identity of the PG core protein is not known, this does not have any impact on the described biochemical properties of the heteromer or its pro-MMP-9 component. It is also shown that a small fraction of the PG, which is not a part of the pro-MMP-9·PG heteromer, can bind gelatin. As for the pro-MMP-9·PG heteromers, this was independent of the CS chains. The structure that mediates the binding of free PG to gelatin is different from the corresponding structure in the pro-MMP-9·PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9·PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.

[Membrane Transport, Structure, Function, and Biogenesis] Cysteine-scanning Analysis of Putative Helix XII in the YgfO Xanthine Permease: ILE-432 AND ASN-430 ARE IMPORTANT

Papakostas, K., Georgopoulou, E., Frillingos, S. — 2008-05-09

Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence ⁴¹⁹ILPASIYVLVENPICAGGLTAILLNIILPGGY⁴⁵⁰ (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80–130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15–40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC₅₀ of 10 µm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.

[Protein Structure and Folding] Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

2008-05-09

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100–5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

[Enzyme Catalysis and Regulation] Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

2008-05-09

Many of the most virulent strains of Salmonella enterica produce two distinct Cu,Zn-superoxide dismutases (SodCI and SodCII). The bacteriophage-encoded SodCI enzyme makes the greater contribution to Salmonella virulence. We have performed a detailed comparison of the functional, structural, and regulatory properties of the Salmonella SodC enzymes. Here we demonstrate that SodCI and SodCII differ with regard to specific activity, protease resistance, metal affinity, and peroxidative activity, with dimeric SodCI exhibiting superior stability and activity. In particular, monomeric SodCII is unable to retain its catalytic copper ion in the absence of zinc. We have also found that SodCI and SodCII are differentially affected by oxygen, zinc availability, and the transcriptional regulator FNR. SodCII is strongly down-regulated under anaerobic conditions and dependent on the high affinity ZnuABC zinc transport system, whereas SodCI accumulation in vitro and within macrophages is FNR-dependent. We have confirmed earlier findings that SodCII accumulation in intracellular Salmonella is negligible, whereas SodCI is strongly up-regulated in macrophages. Our observations demonstrate that differences in expression, activity, and stability help to account for the unique contribution of the bacteriophage-encoded SodCI enzyme to Salmonella virulence.

[Metabolism and Bioenergetics] Hepatocyte Growth Factor Is a Novel Stimulator of Glucose Uptake and Metabolism in Skeletal Muscle Cells

2008-05-09

Skeletal muscle plays a major role in glucose and lipid metabolism. Active hepatocyte growth factor (HGF) is present in the extracellular matrix in skeletal muscle. However, the effects of HGF on glucose and lipid metabolism in skeletal muscle are completely unknown. We therefore examined the effects of HGF on deoxyglucose uptake (DOGU), glucose utilization, and fatty acid oxidation (FAO) in skeletal muscle cells. HGF significantly enhanced DOGU in mouse soleus muscles in vitro. Furthermore, HGF significantly increased: (i) DOGU in a time- and dose-dependent manner; (ii) glucose utilization; and (iii) plasma membrane expression of Glut-1 and Glut-4 in the rat skeletal muscle model of L6 myotubes. HGF-mediated effect on DOGU was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. On the other hand, HGF markedly and significantly decreased FAO in L6 myotubes without affecting the activities of carnitine palmitoyltransferase I and II. Collectively, these results indicate that HGF is a potent activator of glucose transport and metabolism and also a strong inhibitor of FAO in rodent myotubes. HGF, through its ability to stimulate glucose transport and metabolism and to impair FAO, may participate in the regulation of glucose disposal in skeletal muscle.

[Protein Synthesis, Post-Translational Modification, and Degradation] Phosphorylation of MDMX Mediated by Akt Leads to Stabilization and Induces 14-3-3 Binding

Lopez-Pajares, V., Kim, M. M., Yuan, Z.-M. — 2008-05-09

The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser³⁶⁷) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser³⁶⁷. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.

[Mechanisms Of Signal Transduction] Caveolin-1 Up-regulation during Epithelial to Mesenchymal Transition Is Mediated by Focal Adhesion Kinase

Bailey, K. M., Liu, J. — 2008-05-09

Emerging evidence has shown that caveolin-1 is up-regulated in a number of metastatic cancers and can influence various aspects of cell migration. However, in general, the role of caveolin-1 in cancer progression is poorly understood. In the present study, we examined alterations in caveolin-1 expression during epithelial-to-mesenchymal transition (EMT) and the ability of caveolin-1 to alter cancer cell adhesion, an aspect of cell motility. We employed two EMT cell models, the human embryonic carcinoma cell line NT2/D1, and TGF-β1-treated NMuMG cells, which are derived from normal mouse mammary epithelia. Caveolin-1 expression was substantially up-regulated in both cell lines following the induction of EMT and was preceded by increased activation of focal adhesion kinase (FAK) and Src, two known tyrosine kinases involved in EMT. We hypothesized that caveolin-1 expression could be influenced by increased FAK phosphorylation, to which Src is a known contributor. Examination of FAK^+/+ and FAK^-/- mouse embryonic fibroblasts revealed that in cells devoid of FAK, caveolin-1 expression is strikingly diminished. Using FAK and superFAK constructs and the novel FAK inhibitor PF-228, we were able to demonstrate that indeed, FAK can regulate caveolin-1 expression. We also found that Src can contribute to increases in caveolin-1 expression, however, only in the presence of FAK. From the culmination of this data and our functional analyses, we conclude that caveolin-1 expression can be up-regulated during EMT, and further, once expressed, caveolin-1 can greatly influence cancer cell adhesion.

[Transcription, Chromatin, and Epigenetics] An Intramolecular Route for Coupling ATPase Activity in AAA+ Proteins for Transcription Activation

Joly, N., Burrows, P. C., Buck, M. — 2008-05-09

AAA⁺ proteins (ATPases associated with various cellular activities) contribute to many cellular processes and typically function as higher order oligomers permitting the coordination of nucleotide hydrolysis for functional output, which leads to substrate remodeling. The precise mechanisms that enable the relay of nucleotide hydrolysis to their specific functional outputs are largely unknown. Here we use PspF, a specialized AAA⁺ protein required for enhancer-dependent transcription activation in Escherichia coli, as a model system to address this question. We demonstrate that a conserved asparagine is involved in internal organization of the oligomeric ring, regulation of ATPase activity by "trans" factors, and optimizing substrate remodeling. We provide evidence that the spatial relationship between the asparagine residue and the Walker B motif is one key element in the conformational signaling pathway that leads to substrate remodeling. Such functional organization most likely applies to other AAA⁺ proteins, including Ltag (simian virus 40), Rep40 (Adeno-associated virus-2), and p97 (Mus musculus) in which the asparagine to Walker B motif relationship is conserved.

[Mechanisms Of Signal Transduction] Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells

2008-05-09

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

[Enzyme Catalysis and Regulation] Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase

Briggs, L. C., Baldwin, G. S., Miyata, N., Kondo, H., Zhang, X., Freemont, P. S. — 2008-05-09

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPS simultaneously. ATPS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

[Molecular Basis Of Cell and Developmental Biology] Role of Cadherin-mediated Cell-Cell Adhesion in Pancreatic Exocrine-to-Endocrine Transdifferentiation

Minami, K., Okano, H., Okumachi, A., Seino, S. — 2008-05-09

Although pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and β-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic β-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role.

[Protein Structure and Folding] Crystal Structure of a Functional Dimer of the PhoQ Sensor Domain

Cheung, J., Bingman, C. A., Reyngold, M., Hendrickson, W. A., Waldburger, C. D. — 2008-05-09

The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg²⁺, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.

[Protein Synthesis, Post-Translational Modification, and Degradation] Aberrant Folding of Pathogenic Parkin Mutants: AGGREGATION VERSUS DEGRADATION

2008-05-09

Loss-of-function mutations in the Parkin gene (PARK2) are responsible for the majority of autosomal recessive Parkinson disease. A growing body of evidence indicates that misfolding and aggregation of Parkin is a major mechanism of Parkin inactivation, accounting for the loss-of-function phenotype of various pathogenic Parkin mutants. Remarkably, wild-type Parkin is also prone to misfolding under certain cellular conditions, suggesting a more general role of Parkin in the pathogenesis of Parkinson disease. We now show that misfolding of Parkin can lead to two phenotypes: the formation of detergent-insoluble, aggregated Parkin, or destabilization of Parkin resulting in an accelerated proteasomal degradation. By combining two pathogenic Parkin mutations, we could demonstrate that destabilization of Parkin is dominant over the formation of detergent-insoluble Parkin aggregates. Furthermore, a comparative analysis with HHARI, an E3 ubiquitin ligase with an RBR domain highly homologous to that of Parkin, revealed that folding of Parkin is specifically dependent on the integrity of the C-terminal domain, but not on the presence of a putative PDZ-binding motif at the extreme C terminus.

[Dna: Replication, Repair, Recombination, and Chromosome Dynamics] A Model for Oligomeric Regulation of APOBEC3G Cytosine Deaminase-dependent Restriction of HIV

Chelico, L., Sacho, E. J., Erie, D. A., Goodman, M. F. — 2008-05-09

APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C -> U deaminations on single-stranded DNA (ssDNA) with marked 3' -> 5' deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3'-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3'-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5–15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.

[Mechanisms Of Signal Transduction] Insights into GFR{alpha}1 Regulation of Neural Cell Adhesion Molecule (NCAM) Function from Structure-Function Analysis of the NCAM/GFR{alpha}1 Receptor Complex

Sjostrand, D., Ibanez, C. F. — 2008-05-09

The neural cell adhesion molecule NCAM binds glial cell line-derived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFR1. GFR1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFR1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFR1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFR1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFR1. Moreover, although the N-terminal domain of GFR1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFR1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFR1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFR1.

[Mechanisms Of Signal Transduction] cAMP Inhibits Cell Migration by Interfering with Rac-induced Lamellipodium Formation

Chen, L., Zhang, J. J., Huang, X.-Y. — 2008-05-09

Cell migration is critical for animal development and physiological as well as pathological responses. One important step during cell migration is the formation of lamellipodia at the leading edge of migrating cells. Here we report that the second messenger cAMP inhibits the migration of mouse embryonic fibroblast cells and mouse breast tumor cells. cAMP acts downstream of the small GTPase Rac and interferes with the formation of lamellipodia. Moreover, cAMP decreases the phosphorylation of the myosin light chain at the leading edge of cells and increases the phosphorylation of the vasodilator-stimulated phosphoprotein. Together with our previous report of a positive role of another second messenger, cGMP, in lamellipodium formation, our data indicate that cAMP and cGMP play opposite roles in modulating lamellipodium formation.

[Protein Structure and Folding] Nucleation-dependent Tau Filament Formation: THE IMPORTANCE OF DIMERIZATION AND AN ESTIMATION OF ELEMENTARY RATE CONSTANTS

Congdon, E. E., Kim, S., Bonchak, J., Songrug, T., Matzavinos, A., Kuret, J. — 2008-05-09

Filamentous inclusions composed of the microtubule-associated protein tau are found in Alzheimer disease and other tauopathic neurodegenerative diseases, but the mechanisms underlying their formation from full-length protein monomer under physiological conditions are unclear. To address this issue, the fibrillization of recombinant full-length four-repeat human tau was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods and a small-molecule inducer of aggregation, thiazine red. Data were then fit to a simple homogeneous nucleation model with rate constant constraints established from filament dissociation rate, critical concentration, and mass-per-unit length measurements. The model was then tested by comparing the predicted time-dependent evolution of length distributions to experimental data. Results indicated that once assembly-competent conformations were attained, the rate-limiting step in the fibrillization pathway was tau dimer formation. Filament elongation then proceeded by addition of tau monomers to nascent filament ends. Filaments isolated at reaction plateau contained ~2 tau protomers/β-strand spacing on the basis of mass-per-unit length measurements. The model suggests four key steps in the aggregation pathway that must be surmounted for tau filaments to form in disease.

[Molecular Basis Of Cell and Developmental Biology] Polycystic Kidneys Caused by Sustained Expression of Cux1 Isoform p75

2008-05-09

The transcriptional regulator Cux1 (CDP, Cutl1) is aberrantly expressed in mouse models for polycystic kidney disease. Here we show that p75-Cux1, the shortest isoform of Cux1, transcribed from an alternative promoter within intron 20, is also deregulated in polycystic kidneys derived from Pkd1 mutant embryos. To determine the role of the p75-Cux1 isoform in cystogenesis, we generated transgenic mice expressing p75-CUX1 in the kidneys and other tissues. Strikingly, these animals developed polycystic kidneys at variable penetrance and severity, correlating with transgene expression levels. Histological and marker analysis of p75-CUX1-derived polycystic kidneys revealed renal cysts derived from the tubular nephron, supporting a model of autosomal dominant polycystic kidney disease. Transgenic p75-CUX1 kidneys additionally showed an up-regulation of the protooncogene c-myc and a down-regulation of the cyclin-dependent kinase inhibitor p27. Chromatin affinity purification experiments confirmed the direct interaction of Cux1 with the c-myc and p27 promoters. These molecular alterations were accompanied by an increase in cilia length and in the proliferative index of epithelial cells lining the cysts. Together, these results identify an important role for the short isoform of CUX1 in polycystic kidney disease development.

[Transcription, Chromatin, and Epigenetics] Inactivation of NuRD Component Mta2 Causes Abnormal T Cell Activation and Lupus-like Autoimmune Disease in Mice

2008-05-09

Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN- under Th2 activation conditions, but normal levels of IL-4 and IFN- under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN- expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity.

[Molecular Basis Of Cell and Developmental Biology] Transforming Growth Factor-{beta}-stimulated Endocardial Cell Transformation Is Dependent on Par6c Regulation of RhoA

Townsend, T. A., Wrana, J. L., Davis, G. E., Barnett, J. V. — 2008-05-09

Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming growth factor-β (TGFβ) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Recently Par6c (Par6) has been shown to function downstream of TGFβ to recruit Smurf1, an E3 ubiquitin ligase, which targets RhoA for degradation to control apical-basal polarity and tight junction dissolution. We tested the hypothesis that Par6 functions in a pathway that regulates endocardial cell EMT. Here we show that the Type I TGFβ receptor ALK5 is required for endocardial cell EMT. Overexpression of dominant negative Par6 inhibits EMT in AVC endocardial cells, whereas overexpression of wild-type Par6 in normally non-transforming ventricular endocardial cells results in EMT. Overexpression of Smurf1 in ventricular endocardial cells induces EMT. Decreasing RhoA activity using dominant negative RhoA or small interfering RNA in ventricular endocardial cells also increases EMT, whereas overexpression of constitutively active RhoA in AVC endothelial cells blocks EMT. Manipulation of Rac1 or Cdc42 activity is without effect. These data demonstrate a functional role for Par6/Smurf1/RhoA in regulating EMT in endocardial cells.

[Mechanisms Of Signal Transduction] Cardiac Restricted Overexpression of Kinase-dead Mammalian Target of Rapamycin (mTOR) Mutant Impairs the mTOR-mediated Signaling and Cardiac Function

2008-05-09

Mammalian target of rapamycin (mTOR) is a key regulator for cell growth through modulating components of the translation machinery. Previously, numerous pharmacological studies using rapamycin suggested that mTOR has an important role in regulating cardiac hypertrophic growth. To further investigate this assumption, we have generated two lines of cardiac specific mTOR transgenic mice, kinase-dead (kd) mTOR and constitutively active (ca) mTOR, using -myosin heavy chain promoter. -Myosin heavy chain (MHC)-mTOR^kd mice had a near complete inhibition of p70 S6k and 4E-BP1 phosphorylation, whereas MHC-mTOR^ca had a significant increase in p70 S6k and 4E-BP1 phosphorylation. Although the cardiac function of MHC-mTOR^kd mice was significantly altered, the cardiac morphology of these transgenic mice was normal. The cardiac hypertrophic growth in response to physiological and pathological stimuli was not different in MHC-mTOR^kd and MHC-mTOR^ca transgenic mice when compared with that of nontransgenic littermates. These findings suggest that the mTOR-mediated signaling pathway is not essential to cardiac hypertrophic growth but is involved in regulating cardiac function. Additional analysis of cardiac responses to fasting-refeeding or acute insulin administration indicated that MHC-mTOR^kd mice had a largely impaired physiological response to nutrient energy supply and insulin stimulation.

[Rna: Processing and Catalysis] RNaseE and RNA Helicase B Play Central Roles in the Cytoskeletal Organization of the RNA Degradosome

Taghbalout, A., Rothfield, L. — 2008-05-09

The RNA degradosome of Escherichia coli is a multiprotein complex that plays an essential role in normal RNA processing and decay. It was recently shown that the major degradosome constituents are organized in a coiled cytoskeletal-like structure that extends along the length of the cell. Here we show that the endoribonuclease E (RNaseE) and RNA helicase B (RhlB) components of the degradosome can each independently form coiled structures in the absence of the other degradosome proteins. In contrast, the cytoskeletal organization of the other degradosome proteins required the presence of the RNaseE or RhlB coiled elements. Although the RNaseE and RhlB structures were equally competent to support the helical organization of polynucleotide phosphorylase, the cytoskeletal-like organization of enolase occurred only in the presence of the RNaseE coiled structure. The results indicate that the RNA degradosome proteins are components of the bacterial cytoskeleton rather than existing as randomly distributed multiprotein complexes within the cell and suggest a model for the cellular organization of the components within the helical degradosomal structure.

[Mechanisms Of Signal Transduction] Invadopodia and Matrix Degradation, a New Property of Prostate Cancer Cells during Migration and Invasion

Desai, B., Ma, T., Chellaiah, M. A. — 2008-05-09

The present study demonstrated that invadopodia are associated with invasion by degradation of matrix in prostate cancer cells PC3. To find out the presence of invadopodia in PC3 cells, we performed a few comparative analyses with osteoclasts, which utilize podosomes for migration. Our investigations indeed demonstrated that invadopodia are comparable to podosomes in the localization of Wiskott-Aldrich syndrome protein (WASP)/matrix metalloproteinase-9 and the degradation of matrix. Invadopodia are different from podosomes in the localization of actin/vinculin, distribution during migration, and the mode of degradation of extracellular matrix. Invadopodia enable polarized invasion of PC3 cells into the gelatin matrix in a time-dependent manner. Gelatin degradation was confined within the periphery of the cell. Osteoclasts demonstrated directional migration with extensive degradation of matrix underneath and around the osteoclasts. A pathway of degradation of matrix representing a migratory track was observed due to the rearrangement of podosomes as rosettes or clusters at the leading edge. Reducing the matrix metalloproteinase-9 levels by RNA interference inhibited the degradation of matrix but not the formation of podosomes or invadopodia. Competition experiments with TAT-fused WASP peptides suggest that actin polymerization and formation of invadopodia involve the WASP-Arp2/3 complex pathway. Moreover, PC3 cells overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity as compared with PC3 cells and PC3 cells expressing mutant OPN in integrin-binding domain and null for OPN. Thus, we conclude that OPN/integrin vβ3 signaling participates in the process of migration and invasion of PC3 cells through regulating processes essential for the formation and function of invadopodia.

[Protein Structure and Folding] Calcium Ions Are Involved in the Unusual Red Shift of the Light-harvesting 1 Qy Transition of the Core Complex in Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Kimura, Y., Hirano, Y., Yu, L.-J., Suzuki, H., Kobayashi, M., Wang, Z.-Y. — 2008-05-09

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 °C and exhibits an unusual Q_y absorption at 915 nm for the core light-harvesting complex (LH1), an ~35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Q_y transition is strongly dependent on metal cations and Ca²⁺ is involved in the unusual red shift. Removal of the Ca²⁺ resulted in formation of a species with the LH1 Q_y absorption at 880 nm, and addition of the Ca²⁺ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca²⁺ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca²⁺/β-subunit = 1:1 and the binding constant was in 10⁵ m^-1 order of magnitude, which is comparable with those for EF-hand Ca²⁺-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca²⁺ binding were small and occurred slowly, with a typical time constant of ~6 min. Replacement of the Ca²⁺ with other metal cations caused blue shifts of the Q_y bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca²⁺-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the -polypeptide and the N-terminal region of the β-polypeptide.

[Glycobiology and Extracellular Matrices] Targeting of Bone Morphogenetic Protein Growth Factor Complexes to Fibrillin

2008-05-09

Both latent transforming growth factor-β (TGF-β)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-β family of growth factors. Interactions between latent TGF-β-binding protein-1 and TGF-β and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-β family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.

[Protein Structure and Folding] Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

2008-05-09

AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the V_max value for p-methyl red 27-fold with a 3.8-fold increase of the K_m value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The V_max value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

[Protein Structure and Folding] An Oxidized Tryptophan Facilitates Copper Binding in Methylococcus capsulatus-secreted Protein MopE

2008-05-09

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35Å of MopE^* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE^* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE^* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE^*.

[Molecular Basis Of Cell and Developmental Biology] Endothelial Cell Migration in Stable Gradients of Vascular Endothelial Growth Factor A and Fibroblast Growth Factor 2: EFFECTS ON CHEMOTAXIS AND CHEMOKINESIS

2008-05-09

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 µm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

[Protein Structure and Folding] Characterization of the Arabidopsis Heterotrimeric G Protein

Wang, S., Assmann, S. M., Fedoroff, N. V. — 2008-05-09

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the , β, and 1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed G₁ localizes to protoplast membranes, but Gβ exhibits membrane localization only when the G₁ protein is co-overexpressed. Overexpressed G shows membrane localization unaccompanied by overexpression of either Gβ or G₁. We detect fluorescence resonance energy transfer between Gβ and G₁ in the absence of G overexpression and between G and G₁ but only when all three subunits are co-overexpressed. Both G and Gβ are associated with large macromolecular complexes of ~700 kDa in the plasma membrane. G is present in both large complexes and as free G in plasma membranes from wild type plants. In plants homozygous for a null allele of the Gβ gene, G is associated with smaller complexes in the 200–400-kDa range, indicating that its presence in the large complex depends on association with Gβ. Activation of the G subunit with guanosine 5'-3-O-(thio)triphosphate (GTPS) results in partial dissociation of G from the complex. Hydrogen peroxide (H₂O₂) promotes extensive dissociation of the G complex but does not interfere with binding of GTPS to purified recombinant G, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of G itself.

[Mechanisms Of Signal Transduction] Direct Regulation of Genes Involved in Glucose Utilization by the Calcium/Calcineurin Pathway

Ruiz, A., Serrano, R., Arino, J. — 2008-05-09

Failure to use glucose as carbon source results in transcriptional activation of numerous genes whose expression is otherwise repressed. HXT2 encodes a yeast high affinity glucose transporter that is only expressed under conditions of glucose limitation. We show that HXT2 is rapidly and potently induced by environmental alkalinization, and this requires both the Snf1 and the calcineurin pathways. Regulation by calcineurin is mediated by the transcription factor Crz1, which rapidly translocates to the nucleus upon high pH stress, and acts through a previously unnoticed Crz1-binding element (calcineurin-dependent response element) in the HXT2 promoter (-507 GGGGCTG -501). We demonstrate that, in addition to HXT2, many other genes required for adaptation to glucose shortage, such as HXT7, MDH2, or ALD4, transcriptionally respond to calcium and high pH signaling through binding of Crz1 to their promoters. Therefore, calcineurin-dependent transcriptional regulation appears to be a common feature for many genes encoding carbohydrate-metabolizing enzymes. Remarkably, extracellular calcium allows growth of a snf1 mutant on low glucose in a calcineurin/Crz1-dependent manner, indicating that activation of calcineurin is sufficient to override a major deficiency in the glucose-repression pathway. We propose that alkalinization of the medium results in impaired glucose utilization and that activation of certain glucose-metabolizing genes by calcineurin contributes to yeast survival under this stress situation.

[Mechanisms Of Signal Transduction] Activation of KLF8 Transcription by Focal Adhesion Kinase in Human Ovarian Epithelial and Cancer Cells

Wang, X., Urvalek, A. M., Liu, J., Zhao, J. — 2008-05-09

KLF8 (Krüppel-like factor 8) is a transcription factor downstream of focal adhesion kinase (FAK) important in the regulation of the cell cycle and also plays a critical role in oncogenic transformation and epithelial to mesenchymal transition. Here we report the mechanisms by which FAK regulates KLF8 expression in human ovarian epithelial and cancer cells. We show that the overexpression of both KLF8 and FAK in the human ovarian cancer cells as compared with the normal human ovarian surface epithelial cells is critical for cell growth. Using promoter luciferase reporter assays, we demonstrate that exogenous FAK strongly promotes the activity of the KLF8 promoter, and knockdown of FAK inhibits it. KLF8 promoter activity and mRNA levels are induced by expression of constitutively active (CA) phosphatidylinositol 3-kinase (PI3K) or CA-Akt but are repressed by dominant negative Akt or the PI3K inhibitor LY294002. Disruption of an Sp1 binding site in the KLF8 promoter abolishes the FAK- or Sp1-mediated promoter activation. Sp1 knockdown prevents the KLF8 promoter from being activated by Sp1 or CA-Akt, and expression of CA-Akt enhances Sp1 expression in SKOV3ip1 cells. Chromatin immunoprecipitation and oligonucleotide precipitation results show that Sp1 binds to the KLF8 promoter. Taken together, our data suggest that FAK induces KLF8 expression in human ovarian cancer cells by activating the PI3K-Akt signaling pathway, leading to the activation of KLF8 promoter by Sp1.

[Enzyme Catalysis and Regulation] Guinea Pig Chymase Is Leucine-specific: A NOVEL EXAMPLE OF FUNCTIONAL PLASTICITY IN THE CHYMASE/GRANZYME FAMILY OF SERINE PEPTIDASES

2008-05-09

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig -chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe⁸ to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by ₁-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster -chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala²¹⁶ side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.