Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Association of FOXE1 Polyalanine Repeat Region with Papillary Thyroid Cancer

CONTEXT: Polyalanine tract variations in transcription factors have been identified for a wide spectrum of developmental disorders. The thyroid transcription factor forkhead factor E1 (FOXE1) contains a polymorphic polyalanine tract with 12-22 alanines. Single-nucleotide polymorphisms (SNP) close to this locus are associated with papillary thyroid cancer (PTC), and a strong linkage disequilibrium block extends across this region. OBJECTIVE: The objective of the study was to assess whether the FOXE1 polyalanine repeat region was associated with PTC and to assess the effect of polyalanine repeat region variants on protein expression, DNA binding, and transcriptional function on FOXE1-responsive promoters. DESIGN: This was a case-control study. SETTING: The study was conducted at a tertiary referral hospital. PATIENTS AND METHODS: The FOXE1 polyalanine repeat region and tag SNP were genotyped in 70 PTC, with a replication in a further 92 PTC, and compared with genotypes in 5767 healthy controls (including 5667 samples from the Wellcome Trust Case Control Consortium). In vitro studies were performed to examine the protein expression, DNA binding, and transcriptional function for FOXE1 variants of different polyalanine tract lengths. RESULTS: All the genotyped SNP were in tight linkage disequilibrium, including the FOXE1 polyalanine repeat region. We confirmed the strong association of rs1867277 with PTC (overall P = 1 × 10(-7), odds ratio 1.84, confidence interval 1.31-2.57). rs1867277 was in tight linkage disequilibrium with the FOXE1 polyalanine repeat region (r(2) = 0.95). FOXE1(16Ala) was associated with PTC with an odds ratio of 2.23 (confidence interval 1.42-3.50; P = 0.0005). Functional studies in vitro showed that FOXE1(16Ala) was transcriptionally impaired compared with FOXE1(14Ala), which was not due to differences in protein expression or DNA binding. CONCLUSIONS: We have confirmed the previous association of FOXE1 with PTC. Our data suggest that the coding polyalanine expansion in FOXE1 may be responsible for the observed association between FOXE1 and PTC.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Effect of aureomycin on the behaviour of certain free amino acids in oil sardine (Sardinella longiceps) held in ice storage

The course of development of a few free amino acids under the influence of aureomycin in oil sardine (Sardinella lingiceps) held in ice storage was investigated. The levels of leucines and valine regularly increased in the control and aureomycin treated fush throughout the storage period. Alanines and threonine showed similar trend in both control and fish treated with 20ppm aureomycin. These amino acids however showed a gradual fall in fish treated at 5 ppm level. The changes in tyrosine+tryptophane were found to be irregular. Most of the amino acids studied indicated a remarkable change in trend by about the 16th day of ice storage in the case of fish treated with 50ppm aureimycin.

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Mécanismes moléculaires d’activation du récepteur A des peptides natriurétiques

Le récepteur A des peptides natriurétiques (NPRA) fait partie de la famille des guanylates cyclases membranaires. L’activation du NPRA par ses agonistes naturels, ANP et BNP, induit une production de GMPc qui est responsable de leur rôle dans l’homéostasie cardiovasculaire, l’inhibition de l’hypertrophie et de la fibrose cardiaques et la régulation de la lipolyse. Le NPRA est un homodimère non covalent composé d’un domaine extracellulaire de liaison du ligand (ECD), d’un unique domaine transmembranaire (TM), d’un domaine d’homologie aux kinases et d’un domaine guanylate cyclase. Bien que le NPRA ait un rôle physiologique important, les mécanismes moléculaires régissant son processus d’activation restent inconnus. Nous avons donc analysé les premières étapes du processus d’activation du NPRA. Nous avons d'abord étudié le rôle de la dimérisation des ECD dans l’activation du récepteur. Nous avons utilisé les techniques de liaison de radioligand, de FRET et de modélisation moléculaire, pour caractériser la liaison à l’ECD des agonistes naturels, d’un superagoniste et d’un antagoniste. L’ANP se lie à un dimère d’ECD préformé et la dimérisation spontanée est l’étape limitante du processus de liaison. De plus, comme le démontrent nos études de FRET, tous les peptides, incluant l’antagoniste, stabilisent le récepteur sous sa forme dimérique. Cependant, l’antagoniste A71915 stabilise le dimère d’ECD dans une conformation différente de celle induite par l’ANP. La dimérisation du NPRA semble donc nécessaire, mais non suffisante à l’activation du récepteur. L’état d’activation du NPRA dépend plutôt de l’orientation des sous unités dans le dimère. Nous avons ensuite étudié le mécanisme moléculaire de transduction du signal à travers la membrane. Plusieurs études ont suggéré que l’activation du NPRA implique un changement de conformation du domaine juxtamembranaire (JM). Cependant, les études de cristallographie de l’ECD soluble de NPRA n’ont pas permis de documenter la structure du JM et le changement de conformation impliqué dans la transduction du signal reste inconnu. Pour analyser ce changement de conformation, nous avons d’abord séquentiellement substitué les neuf acides aminés du JM par une cystéine. En étudiant la capacité des mutants à former des dimères covalents de façon constitutive ou induite par l’ANP, nous avons pu évaluer la proximité relative des résidus du JM, avant et après activation du NPRA. Ces résultats ont démontré la proximité élevée de certains résidus spécifiques et sont en contradiction avec les données cristallographiques. Nous avons également démontré que le domaine intracellulaire impose une contrainte conformationnelle au JM à l’état de base, qui est levée après liaison de l’ANP. En introduisant de 1 à 5 alanines dans l’hélice-α transmembranaire, nous avons montré qu’une rotation des TM de 40° induit une activation constitutive du NPRA. Le signal d’activation pourrait donc être transmis à travers la membrane par un mécanisme de rotation des TM. En utilisant nos données expérimentales, nous avons généré le premier modèle moléculaire illustrant la conformation active du NPRA, où les domaines JM et TM sont représentés. Dans son ensemble, cette étude apporte une meilleure compréhension des mécanismes moléculaires régissant les premières étapes du processus complexe d’activation du NPRA.

Couplage du récepteur à sept domaines transmembranaires GABA-B1 aux voies intracellulaires de signalisation en absence de GABA-B2

Le GABA est le principal neurotransmetteur inhibiteur du SNC et est impliqué dans le développement du cerveau, la plasticité synaptique et la pathogénèse de maladies telles que l’épilepsie, les troubles de l’anxiété et la douleur chronique. Le modèle actuel de fonctionnement du récepteur GABA-B implique l’hétérodimérisation GABA-B1/B2, laquelle est requise au ciblage à la surface membranaire et au couplage des effecteurs. Il y est cependant des régions du cerveau, des types cellulaires et des périodes du développement cérébral où la sous-unité GABA-B1 est exprimée en plus grande quantité que GABA-B2, ce qui suggère qu’elle puisse être fonctionnelle seule ou en association avec des partenaires inconnus, à la surface cellulaire ou sur la membrane réticulaire. Dans le cadre de cette thèse, nous montrons la capacité des récepteurs GABA-B1 endogènes à activer la voie MAPK-ERK1/2 dans la lignée dérivée de la glie DI-TNC1, qui n’exprime pas GABA-B2. Les mécanismes qui sous-tendent ce couplage demeurent mal définis mais dépendent de Gi/o et PKC. L’immunohistochimie de récepteurs endogènes montre par ailleurs que des anticorps GABA-B1 dirigés contre la partie N-terminale reconnaissent des protéines localisées au RE tandis des anticorps C-terminaux (CT) marquent une protéine intranucléaire. Ces données suggèrent que le domaine CT de GABA-B1 pourrait être relâché par protéolyse. L’intensité des fragments potentiels est affectée par le traitement agoniste tant en immunohistochimie qu’en immunobuvardage de type western. Nous avons ensuite examiné la régulation du clivage par le protéasome en traitant les cellules avec l’inhibiteur epoxomicine pendant 12 h. Cela a résulté en l’augmentation du marquage intranucléaire de GABA-B1-CT et d’un interacteur connu, le facteur de transcription pro-survie ATF-4. Dans des cellules surexprimant GABA-B1-CT, l’induction et la translocation nucléaire d’ATF-4, qui suit le traitement epoxomicine, a complètement été abolie. Cette observation est associée à une forte diminution du décompte cellulaire. Étant donné que les trois derniers résidus de GABA-B1-CT (LYK) codent un ligand pseudo-PDZ et que les protéines à domaines PDZ sont impliquées dans la régulation du ciblage nucléaire et de la stabilité de protéines, en complément de leur rôle d’échaffaud à la surface cellulaire, nous avons muté les trois derniers résidus de GABA-B1-CT en alanines. Cette mutation a complètement annulé les effets de GABA-B1-CT sur l’induction d’ATF-4 et le décompte cellulaire. Cette deuxième série d’expériences suggère l’existence possible de fragments GABA-B1 intranucléaires régulés par le traitement agoniste et le protéasome dans les cellules DI-TNC1. Cette régulation d’ATF-4 dépend des résidus LYK de GABA-B1-CT, qui modulent la stabilité de GABA-B1-CT et favorisent peut-être la formation d’un complexe multiprotéique incluant GABA-B1-CT, ATF-4, de même qu’une protéine d’échaffaudage inconnue. En somme, nous démontrons que les sous-unités GABA-B1 localisées au RE, lorsque non-hétérodimérisées avec GABA-B2, demeurent capables de moduler les voies de signalisation de la prolifération, la différentiation et de la survie cellulaire, via le couplage de protéines G et possiblement la protéolyse régulée. Les mécanismes de signalisation proposés pourraient servir de nouvelle plate-forme dans la compréhension des actions retardées résultant de l’activation des récepteurs 7-TMs.

Importance de la phosphorylation de la ligase Itch dans la reconnaissance et l'ubiquitylation des protéines à domaine SH3

Itch est une ligase de l’ubiquitine impliquée dans la reconnaissance et la dégradation des protéines par le protéasome. Itch contient trois sites phosphorylés par JNK et il a été démontré que la phosphorylation de ces résidus est nécessaire pour que Itch puisse reconnaître et ubiquityler les protéines c-Jun et JunB. Ces sites de phosphorylation se retrouvent dans le domaine PRD responsable des interactions de Itch avec les protéines à domaine SH3. Si la phosphorylation de Itch par JNK est importante pour réguler son activité avec c-Jun et JunB, on connaît peu de choses sur les interactions de Itch avec les protéines à domaine SH3 ainsi que l’implication de la phosphorylation dans leur régulation. Nous avons donc créé des mutants de Itch par mutagenèse dirigée où les sites de phosphorylation étaient remplacés par des alanines (mutant non phosphorylable) et où l’un des trois sites était remplacé par un acide aspartique (mutant constitutivement phosphorylé). Ces mutants sont utilisés dans des tests d’interaction et d’ubiquitylation, dans le but de déterminer l’impact de la phosphorylation de Itch dans la reconnaissance et l’ubiquitylation des protéines SH3. Nos résultats montrent que, contrairement au modèle proposé, la phosphorylation de Itch n’est pas essentielle à l’interaction de Itch avec l’endophiline, mais la phosphorylation de Itch module l’ubiquitylation ainsi que la dégradation de l’endophiline. La régulation de l’interaction de Itch avec ses substrats est donc différente selon le substrat.

Biology and characterisation of polyalanine as an emerging pathological marker

Dix-huit maladies humaines graves ont jusqu'ici été associées avec des expansions de trinucléotides répétés (TNR) codant soit pour des polyalanines (codées par des codons GCN répétés) soit pour des polyglutamines (codées par des codons CAG répétés) dans des protéines spécifiques. Parmi eux, la dystrophie musculaire oculopharyngée (DMOP), l’Ataxie spinocérébelleuse de type 3 (SCA3) et la maladie de Huntington (MH) sont des troubles à transmission autosomale dominante et à apparition tardive, caractérisés par la présence d'inclusions intranucléaires (IIN). Nous avons déjà identifié la mutation responsable de la DMOP comme étant une petite expansion (2 à 7 répétitions supplémentaires) du codon GCG répété du gène PABPN1. En outre, nous-mêmes ainsi que d’autres chercheurs avons identifié la présence d’événements de décalage du cadre de lecture ribosomique de -1 au niveau des codons répétés CAG des gènes ATXN3 (SCA3) et HTT (MH), entraînant ainsi la traduction de codons répétés hybrides CAG/GCA et la production d'un peptide contenant des polyalanines. Or, les données observées dans la DMOP suggèrent que la toxicité induite par les polyalanines est très sensible à leur quantité et leur longueur. Pour valider notre hypothèse de décalage du cadre de lecture dans le gène ATXN3 dans des modèles animaux, nous avons essayé de reproduire nos constatations chez la drosophile et dans des neurones de mammifères. Nos résultats montrent que l'expression transgénique de codons répétés CAG élargis dans l’ADNc de ATXN3 conduit aux événements de décalage du cadre de lecture -1, et que ces événements sont néfastes. À l'inverse, l'expression transgénique de codons répétés CAA (codant pour les polyglutamines) élargis dans l’ADNc de ATXN3 ne conduit pas aux événements de décalage du cadre de lecture -1, et n’est pas toxique. Par ailleurs, l’ARNm des codons répétés CAG élargis dans ATXN3 ne contribue pas à la toxicité observée dans nos modèles. Ces observations indiquent que l’expansion de polyglutamines dans nos modèles drosophile et de neurones de mammifères pour SCA3 ne suffit pas au développement d'un phénotype. Par conséquent, nous proposons que le décalage du cadre de lecture ribosomique -1 contribue à la toxicité associée aux répétitions CAG dans le gène ATXN3. Pour étudier le décalage du cadre de lecture -1 dans les maladies à expansion de trinucléotides CAG en général, nous avons voulu créer un anticorps capable de détecter le produit présentant ce décalage. Nous rapportons ici la caractérisation d’un anticorps polyclonal qui reconnaît sélectivement les expansions pathologiques de polyalanines dans la protéine PABPN1 impliquée dans la DMOP. En outre, notre anticorps détecte également la présence de protéines contenant des alanines dans les inclusions intranucléaires (IIN) des échantillons de patients SCA3 et MD.

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence A beta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63 +/- 18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet. (C) 2008 Elsevier B.V. All rights reserved.

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Ionizing radiation and short wavelength UV activate NF-κB through two distinct mechanisms

We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and γ radiation, activate transcription factor NF-κB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-κB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-κB inhibitor IκBα. In both cases, induction of NF-κB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and γ radiation induce degradation of IκBs by means of the ubiquitin/proteasome pathway. However, although the induction of IκBα degradation by γ rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IκBα degradation was not dependent on phosphorylation of these residues. Even the “super repressor” IκBα mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that γ radiation led to activation of IKK, the protein kinase that phosphorylates IκBα at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKβ mutant prevented NF-κB activation by γ radiation, but not by UV-C. These results indicate that γ radiation and UV-C activate NF-κB through two distinct mechanisms.

A Modulatory Role for Clathrin Light Chain Phosphorylation in Golgi Membrane Protein Localization during Vegetative Growth and during the Mating Response of Saccharomyces cerevisiae

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the α-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.

Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-α-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2−/− T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2−/− T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.