The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Plasma Kallikrein and Angiotensin I-converting enzyme N- and C-terminal domain activities are modulated by the insertion/deletion polymorphism

Angiotensin I-converting enzyme (ACE) is recognized as one of the main effector molecules involved in blood pressure regulation. In the last few years some polymorphisms of ACE such as the insertion/deletion (I/D) polymorphism have been described, but their physiologic relevance is poorly understood. In addition, few studies investigated if the specific activity of ACE domain is related to the I/D polymorphism and if it can affect other systems. The aim of this study was to establish a biochemical and functional characterization of the I/D polymorphism and correlate this with the corresponding ACE activity. For this purpose, 119 male brazilian army recruits were genotyped and their ACE plasma activities evaluated from the C- and N-terminal catalytic domains using fluorescence resonance energy transfer (FRET) peptides, specific for the C-domain (Abz-LFK(Dnp)OH), N-domain (Abz-SDK(Dnp)P-OH) and both C- and N-domains (Abz-FRK(Dnp)P-OH). Plasma kallikrein activity was measured using Z-Phe-Arg-AMC as substrate and inhibited by selective plasma kallikrein inhibitor (PKSI). Some physiological parameters previously described related to the I/D polymorphism such as handgrip strength, blood pressure, heart rate and BMI were also evaluated. The genotype distribution was II n = 27, ID n = 64 and DD n = 28. Total plasma ACE activity of both domains in II individuals was significantly lower in comparison to ID and DD. This pattern was also observed for C- and N-domain activities. Difference between ID and DD subjects was observed only with the N-domain specific substrate. Blood pressure, heart rate, handgrip strength and BMI were similar among the genotypes. This polymorphism also affected the plasma kallikrein activity and DD group presents high activity level. Thus, our data demonstrate that the I/D ACE polymorphism affects differently both ACE domains without effects on handgrip strength. Moreover, this polymorphism influences the kallikrein-kinin system of normotensive individuals. (C) 2009 Elsevier Ltd. All rights reserved.

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.

The Interaction of the Calcium Sensitiser Levosimendan with Cardiac Troponin C

Cardiac failure is one of the leading causes of mortality in developed countries. As life expectancies of the populations of these countries grow, the number of patients suffering from cardiac insufficiency also increase. Effective treatments including the use of calcium sensitisers are being sought. They cause a positive inodilatory effect on cardio-myocytes without deleterious effects (arrhythmias) resulting from increases in intracellular calcium concentration. Levosimendan is a novel calcium sensitiser that hasbeen proved to be a welltolerated and effective treatment for patients with severe decompensated heart failure. Cardiac troponin C (cTnC) is its target protein. However, there have been controversies about the interactions between levosimendan and cTnC. Some of these controversies have been addressed in this dissertation. Furthermore, studies on the calcium sensitising mechanism based on the interactions between levosimendan and cTnC as followed by nuclear magnetic resonance(NMR) are presented and discussed. Levosimendan was found to interact with bothdomains of the calcium-saturated cTnC in the absence of cardiac troponin I (cTnI). In the presence of cTnI, the C-domain binding site was blocked and levosimendan interacted only with the regulatory domain of cTnC. This interaction may have caused the observed calcium sensitising effect by priming the N-domain for cTnI binding thereby extending the lifetime of that complex. It is suggested that this is achieved by shifting the equilibrium between open and closed conformations.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B.subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ∼0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (∼0.9 min–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (∼40 min–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Analysis of the mechanisms controlling the activity of flp1, a chromosomal passenger protein in the fission Schizosaccharomyces pombe

ABSTRACT In S. cerevisiae, the protein phosphatase Cdc14pwt is essential far mitotic exit through its contribution to reducing mitotic CDK activity. But Cdc14pwt also acts as a mare general temporal coordinator of mid and late mitotic events by controlling the partitioning of DNA, microtubule stability and cytokinesis. Cdc14pwt orthologs are well conserved from yeasts to humans, and sequence comparison revealed the presence of three domains, A, B and C, of which A and B form the catalytic domain. Cdc14pwt orthologs are regulated (in part) through cell cycle dependent changes in their localization. Some of them are thought to be kept inactive by sequestration in the nucleolus during interphase. This is the case for flp1pwt, the single identified Cdc14pwt ortholog in the fission yeast S. pombe. In early mitosis, flp1pwt leaves the nucleolus and localizes to the kinetochores, the contractile ring and the mitotic spindle, suggesting that it has multiple substrates and regulates many mitotic processes. flp1D cells show a high chromosome loss rate and septation defects, suggesting a role for flp1wt in the fidelity of chromosome transmission and cytokinesis. The aim of this study is to characterize the mechanisms underlying flp1pwt functions and the control of its activity. A structure-function analysis has revealed that the presence of both A and B domains is required for biological function and for proper flp1pwt mitotic localization. In contrast, the C domain of flp1pwt is responsible for its proper nucleolar localization in G2/interphase. My data suggest that dephosphorylation of substrates by flp1pwt is not necessary for any changes in localization of flp1pwt except that at the medial ring. In that particular case, the catalytic activity of flp1pwt is required for efficient localization, therefore revealing an additional level of regulation. All the functions of flp1pwt assayed to date require its catalytic activity, emphasizing the importance of further identification of its substrates. As described for other orthologs, the capability of selfinteraction and phosphorylation status might help to control flp1pwt activity. My data suggest that flp1pwt forms oligomers in vivo and that phosphorylation is not essential far localization changes of the protein. In addition, the hypophosphorylated form of flp1pwt might be specifically involved in the promotion of cytokinesis. The results of this study suggest that multiple modes of regulation including localization, selfassociation and phosphorylation allow a fine-tuning regulation of flp1pwt phosphatase activity, and more generally that of Cdc14pwt family of phosphatases. RESUME Chez la levure S. cerevisiae, la protéine phosphatase Cdc14pwt est essentielle pour la sortie de mitose du fait de sa contribution dans la réduction d'activité des CDK mitotiques. Comme elle contrôle également le partage de l'ADN, la stabilité des microtubules et la cytokinèse, Cdc14pwt est en fait considérée comme un coordinateur temporel général des évènements de milieu et de fin de mitose. Les orthologues de Cdc14pwt sont bien conservés, des levures jusqu'à l'espèce humaine. Des comparaisons de séquence ont révélé la présence de trois domaines A, B et C, les deux premiers constituant le domaine catalytique. Ils sont régulés (en partie) via des changements dans leur localisation, eux-mêmes dépendants du cycle cellulaire. Plusieurs de ces orthologues sont supposés inactivés par séquestration dans le nucléole en interphase, ce qui est le cas de flp1pwt le seul orthologue de Cdc14pwt identifié chez la levure fissipare S, pombe. En début de mitose, flp1pwt quitte le nucléole et localise au niveau des kinetochores, de l'anneau contractile d'actine et du fuseau mitotique, ce qui laisse supposer de multiples substrats et fonctions. Comme les cellules délétées pour le gène flp1wt présentent un taux élevé de perte de chromosome et des défauts de septation, flp1pwt semble jouer un rôle dans la fidélité de la transmission du matériel génétique et la cytokinèse. Le but de cette étude est de caractériser les mécanismes impliqués dans les fonctions assurées par flp1pwt d'une part, et dans le contrôle de son activité d'autre part. Une analyse structure-fonction a révélé que la présence simultanée des deux domaines A et B est requise pour la fonction biologique de flp1pwt et sa localisation correcte pendant la mitose. Par contre, le domaine C de flp1pwt confère une localisation nucléolaire adéquate en G2/interphase. Mes données suggèrent que la déphosphorylation de substrats par flp1pwt est dispensable pour sa localisation correcte excepté celle à l'anneau médian, qui requiert dans ce cas, l'activité catalytique de flp1pwt, révélant ainsi un niveau de régulation supplémentaire. Toutes les fonctions de flp1 pwt testées jusqu'à présent nécessitent également son activité catalytique, ce qui accentue l'importance de l'identification future de ses substrats. Comme cela a déjà été décrit pour d'autres orthologues, la capacité d'auto-intéraction et le niveau de phosphorylation pourraient contrôler l'activité de flp1pwt. En effet, mes données suggèrent que flp1pwt forme des oligomères in vivo et que la phosphorylation n'est pas essentielle pour les changements de localisation observés pour la protéine. De plus, la forme hypophosphorylée de flp1pwt pourrait être spécifiquement impliquée dans la promotion de la cytokinèse. De multiples modes de régulation incluant la localisation, l'auto-association et la phosphorylation semblent permettre un contrôle fin et subtil de l'activité de la phosphatase flp1pwt, et plus généralement celle des protéines de la famille de Cdc14pwt.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei

ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.

Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure–function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al3+ sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure–function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al3+ sensing.