Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Phosphoinositide 3-kinases in lysophosphatidic acid signaling: regulation and cross-talk with the Ras/mitogen-activated protein kinase pathway

Recent reports have shown that phosphoinositide 3-kinases (PI3Ks) mediate various biological activities of lysophosphatidic acid (LPA), including cell proliferation or survival. In addition, these enzymes have been proposed to be early intermediates of mitogen-activated protein kinase (MAPK) activation. Here we summarize our current knowledge of the mechanisms underlying these observations. p110gamma is an isoform of PI3K that can be activated in vitro by Gbetagamma subunits and was therefore considered as the logical candidate to mediate responses induced by G protein-coupled receptor (GPCR) agonists. In agreement with this, p110gamma has been involved in different biochemical models linking Gbetagamma to MAPK activation. Nevertheless, its apparent tissue-specific distribution has raised questions regarding the physiological relevance of these models. In addition, LPA can activate p110beta, a member of the phosphotyrosine-dependent PI3K subfamily that participates in the mitogenic effect of LPA. Its activation is thought to involve a synergistic effect of Gbetagamma and phosphotyrosine motifs provided by a transactivated EGF receptor/Gab1 pathway. We are currently studying a possible role of p110beta upstream from Ras, suggesting that this protein could provide a novel connection between betagamma and the MAPK pathway.     

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.     

Structural basis for Chk1 inhibition by UCN-01

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.     

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.     

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.     

Pulsatile urea excretion in the gulf toadfish: mechanisms and controls

Opsanus beta expresses a full complement of ornithine–urea cycle (OUC) enzymes and is facultatively ureotelic, reducing ammonia-N excretion and maintaining urea-N excretion under conditions of crowding/confinement. The switch to ureotelism is keyed by a modest rise in cortisol associated with a substantial increase in cytosolic glutamine synthetase for trapping of ammonia-N and an upregulation of the capacity of the mitochondrial OUC to use glutamine-N. The entire day's urea-N production is excreted in 1 or 2 short-lasting pulses, which occur exclusively through the gills. The pulse event is not triggered by an internal urea-N threshold, is not due to pulsatile urea-N production, but reflects pulsatile activation of a specific branchial excretion mechanism that rapidly clears urea-N from the body fluids. A bidirectional facilitated diffusion transporter, with pharmacological similarity to the UT-A type transporters of the mammalian kidney, is activated in the gills, associated with an increased trafficking of dense-cored vesicles in the pavement cells. An 1814 kB cDNA (‘tUT’) coding for a 475–amino acid protein with approximately 62% homology to mammalian UT-A's has been cloned and facilitates phloretin-sensitive urea transport when expressed in Xenopus oocytes. tUT occurs only in gill tissue, but tUT mRNA levels do not change over the pulse cycle, suggesting that tUT regulation occurs at a level beyond mRNA. Circulating cortisol levels consistently decline prior to a pulse event and rise thereafter. When cortisol is experimentally clamped at high levels, natural pulse events are suppressed in size but not in frequency, an effect mediated through glucocorticoid receptors. The cortisol decline appears to be permissive, rather than the actual trigger of the pulse event. Fluctuations in circulating AVT levels do not correlate with pulses; and injections of AVT (at supraphysiological levels) elicit only minute urea-N pulses. However, circulating 5-hydroxytryptamine (5-HT) levels fluctuate considerably and physiological doses of 5-HT cause large urea-N pulse events. When the efferent cranial nerves to the gills are sectioned, natural urea pulse events persist, suggesting that direct motor output from the CNS to the gill is not the proximate control.     

Identification of Wolbachia--host interacting factors through cytological analysis

Manipulation of host reproduction and efficient maternal transmission have facilitated the global spread of Wolbachia through millions of insect species. Cytological studies of the most common Wolbachia-induced phenotype, cytoplasmic incompatibility (CI), demonstrate that Wolbachia induce CI by altering host cell cycle timing. Cytological analyses also suggest that microtubules and motor proteins may play a role in the maternal and somatic transmission of Wolbachia.