Institute of Biochemistry

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

Osteoporosis is a bone disorder with remarkable changes in bone biologic material and consequent bone structural distraction, affecting millions of people around the world from different ethnic groups. Bone fragility is the worse outcome of the disease, which needs long term therapy and medical management, especially in the elderly. Many involved genes including environmental factors have been introduced as the disease risk factors so far, of which genes should be considered as effective early diagnosis biomarkers, especially for the individuals from high-risk families. In this review, a number of important criteria involved in osteoporosis are addressed and discussed.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.

ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

Chaperones (stress proteins) are essential proteins to help the formation and maintenance of the proper conformation of other proteins and to promote cell survival after a large variety of environmental stresses. Therefore, normal chaperone function is a key factor for endogenous stress adaptation of several tissues. However, altered chaperone function has been associated with the development of several diseases; therefore, modulators of chaperone activities became a new and emerging field of drug development. Inhibition of the 90 kDa heat shock protein (Hsp)90 recently emerged as a very promising tool to combat various forms of cancer. On the other hand, the induction of the 70 kDa Hsp70 has been proved to be an efficient help in the recovery from a large number of diseases, such as, for example, ischemic heart disease, diabetes and neurodegeneration. Development of membrane-interacting drugs to modify specific membrane domains, thereby modulating heat shock response, may be of considerable therapeutic benefit as well. In this review, we give an overview of the therapeutic approaches and list some of the key questions of drug development in this novel and promising therapeutic approach.

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large-scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole-genome sequencing of laboratory-evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton-motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF-dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade-offs under antibiotic selection.

Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

Thermal stress in living cells produces multiple changes that ultimately affect membrane structure and function. We report that two members of the family of small heat-shock proteins (sHsp) (alpha-crystallin and Synechocystis HSP17) have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids. In anionic membranes of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine, both HSP17 and alpha-crystallin strongly stabilize the liquid-crystalline state. Evidence from infrared spectroscopy indicates that lipid/sHsp interactions are mediated by the polar headgroup region and that the proteins strongly affect the hydrophobic core. In membranes composed of the nonbilayer lipid dielaidoylphosphatidylethanolamine, both HSP17 and alpha-crystallin inhibit the formation of inverted hexagonal structure and stabilize the bilayer liquid-crystalline state, suggesting that sHsps can modulate membrane lipid polymorphism. In membranes composed of monogalactosyldiacylglycerol and phosphatidylglycerol (both enriched with unsaturated fatty acids) isolated from Synechocystis thylakoids, HSP17 and alpha-crystallin increase the molecular order in the fluid-like state. The data show that the nature of sHsp/membrane interactions depends on the lipid composition and extent of lipid unsaturation, and that sHsps can regulate membrane fluidity. We infer from these results that the association between sHsps and membranes may constitute a general mechanism that preserves membrane integrity during thermal fluctuations.

Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [35S]GTPγS assays at the mu opioid receptor but failed to recruit β-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers.

Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK. Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK. Mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; MAPKK, MAPK kinase. cascades control the activity of three sets of effector protein kinases (extracellular signal-regulated protein kinases (ERKs), Jun kinases (JNKs), and p38s). The central element in each MAPK pathway is a module of three protein kinases, MAPKK kinase, MAPKK, and MAPK (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1136) Google Scholar). The three sets of effector MAPK differ in type of activating stimulus: JNKs and p38/HOG-1 primarily respond to stress (e.g. heat shock), and ERKs primarily respond to mitogens. However, a stimulus can activate more than one class of MAPK; the contribution of each pathway is cell type-dependent, and MAPK pathways can both synergize and antagonize. This is caused in part by regulatory proteins influencing signaling by a range of mechanisms including scaffolding (e.g. JIP-1, STE5), regulating localization (e.g. Ksr), or recruitment to targets (e.g. 14-3-3 proteins) (2Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 3Roulston A. Reinhard C. Amiri P. Williams L.T. J. Biol. Chem. 1998; 273: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 4Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar). Here we describe a novel family of MAPK control proteins, homologues of fly tribbles. Drosophila tribbles was shown to regulate String activity and hence mitosis during ventral furrow formation (5Grosshans J. Wieschaus E. Cell. 2000; 101: 523-531Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 6Rorth P. Szabo K. Texido G. Mol. Cell. 2000; 6: 23-30Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 7Seher T.C. Leptin M. Curr. Biol. 2000; 10: 623-629Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 8Mata J. Curado S. Ephrussi A. Rorth P. Cell. 2000; 101: 511-522Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). A canine Trb-2-like protein has been described in the literature as a transiently expressed, mitogen induced, and highly labile cytoplasmic phosphoprotein, but its biological function was not characterized (9Wilkin F. Suarez-Huerta N. Robaye B. Peetermans J. Libert F. Dumont J.E. Maenhaut C. Eur. J. Biochem. 1997; 248: 660-668Crossref PubMed Scopus (52) Google Scholar, 10Wilkin F. Savonet V. Radulescu A. Petermans J. Dumont J.E. Maenhaut C. J. Biol. Chem. 1996; 271: 28451-28457Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Rat Trb was shown to be rapidly upregulated during neuronal cell apoptosis (11Mayumi-Matsuda K. Kojima S. Suzuki H. Sakata T. Biochem. Biophys. Res. Commun. 1999; 258: 260-264Crossref PubMed Scopus (61) Google Scholar). Recently Trb-3 has been reported to regulate Akt activation in liver by insulin (12Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (716) Google Scholar) and regulate ATF4 activity (13Ord D. Ord T. Exp. Cell Res. 2003; 286: 308-320Crossref PubMed Scopus (76) Google Scholar, 14Bowers A.J. Scully S. Boylan J.F. Oncogene. 2003; 22: 2823-2835Crossref PubMed Scopus (127) Google Scholar). We show here that Trbs bind to MAPKK and regulate MAPK activation suggesting that Trb function may be broader than reported previously. Plasmids—FLAG-MKK7 (15Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), FLAG-MEK-1 (16Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (228) Google Scholar), IL-8-luc (17Wyllie D.H. Kiss-Toth E. Smith S.C. Visintin A. Boussouf S. Segal D. Duff G.W. Dower S.K. J. Immunol. 2000; 165: 7125-7132Crossref PubMed Scopus (234) Google Scholar), and LHRE-TK-luc (18Maamra M. Finidori J. Von Laue S. Simon S. Justice S. Webster J. Dower S. Ross R. J. Biol. Chem. 1999; 274: 14791-14798Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) were described earlier. V12 Ras was a gift of J. Downward. pAP-1 luc, pNFκB luc, pFR luc, pFA-CHOP, pFA2-Elk-1, pMEKK-1 pMEK-1, and pMEK-3 were part of the PathDetect system (Stratagene). Quantitative real time-PCR was performed to characterize the expression profile of human tribbles genes by using the Human Rapid-Scan panel (Origene). MRNA levels are expressed as relative units normalized for glyceraldehyde-3-phosphate dehydrogenase expression. Cell Cultures and Transfections—HeLa (ECACC, 85060701) and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin-streptomycin. Raw cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin-streptomycin. Cells (1.5 × 104 per well) were seeded into 96-well tissue culture plates 24 h prior to transfection. Transfections were performed using SuperFect (Qiagen) according to the manufacturer's advice; each well received 500 ng of inducible reporter construct (pIL-8 luc, pAP-1 luc, pNFκB luc, or pLHRE-TK luc), 100 ng of pTK-RLuc (Promega) for normalization of transfection efficiency, and 50 ng of htrb-1 or htrb-3 expression vectors under investigation unless stated otherwise in the appropriate figure legend. 500 ng of pFR luc, 100 ng of pTK-RLuc, and 10 ng of pFA-CHOP or pFA2-Elk-1, 25 ng pMEK-1, or pMEK-3 plasmids were transfected to specifically activate p38 or ERK and to study the effect of htrb-3 on the activation. Sufficient pCDNA3.1 (Invitrogen) (“empty vector”) was added to keep the total DNA dose constant at 700 ng/well. 2 h after transfection, cells were washed, and 100 μl of fresh medium was added. Triplicate wells were transfected for each treatment. Stimulations were performed for 4 h (unless indicated otherwise) 24 h later. 2 nm IL-1β or 10 ng/ml tumor necrosis factor α, 0.5 μg/ml human growth hormone, 50 ng/ml PMA, or 10 nm of the other cytokines listed on Supplementary Table I was used (unless stated otherwise on the figure). Agonists were prepared and added as 10× stocks in 11 μl of phosphate-buffered saline. Reporter levels were measured following 4 h of stimulation using the dual-luciferase system (Promega) as recommended by the manufacturer. Cytokines—IL-β was a kind gift from the Immunex Corporation. The other human cytokine preparations were kindly provided by Dr. Steve Poole, National Institute for Biological Standards Control. Western Blotting—For detection of pJun, pERK, and pMKK4, polyclonal antibodies were purchased from Sigma. Protein concentrations of cell lysates were determined, and an equal amount of total protein was loaded in each lane. Kinase assays were performed by using the appropriate kits from New England Biolabs. Human tribbles Identification and Sequence—Using a transcription expression screen for gene products mediating inflammatory cytokine signaling (19Kiss-Toth E. Dower S.K. Sayers J.R. Anal. Biochem. 2001; 288: 230-232Crossref PubMed Scopus (8) Google Scholar, 20Kiss-Toth E. Guesdon F.M.J. Wyllie D.H. Qwarnstrom E.E. Dower S.K. J. Immunol. Methods. 2000; 239: 125-135Crossref PubMed Scopus (25) Google Scholar, 21Dower S.K. Kiss-Toth E. Curr. Genomics. 2002; 3: 131-137Crossref Scopus (3) Google Scholar), we identified a human tribbles homologue (htrb-1) which regulated the human IL-8 promoter in HeLa cells. The library clone encoded a portion of the 3′-untranslated region extending to the poly(A) tail (the library was oligo(dT)-primed) transcribed in the expression vector (pCDM8) in the sense orientation. PCR experiments using coding region primers to detect endogenous trb-1 message but not the truncated noncoding library-derived transcript showed that transfection of the library clone into HeLa cells caused a >10-fold increase in the level of endogenous trb-1 message, which correlated with inhibition of IL-8 promoter activity. 2E. Kiss-Toth, D. H. Wyllie, V. Jozsa, K. M. Oxley, T. Polgar, E. E. Qwarnstrom, and S. K. Dower, submitted for publication. Two other human tribbles homologues, htrb-2 and htrb-3 (41 and 51% identical to Htrb-1, respectively), were identified by data base searching; sequences have been submitted to GenBank™ (accession numbers AF250310 and AF250311). A family comprised of three trbs is found in mammals, and orthologs have been identified in both vertebrates and invertebrates. 3E. Kiss-Toth and S. K. Dower, unpublished data. All three Trb proteins share a central Trb domain. In addition, each has N-terminal (70–100 residues) and C-terminal (∼25 residues) domains, which are neither closely related to any other sequences nor closely related to each other. The Trb domain is homologous to protein serine-threonine kinases but lacks the active site lysine and is predicted to be kinase dead as shown for canine Trb-2 and Tribbles (5Grosshans J. Wieschaus E. Cell. 2000; 101: 523-531Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 9Wilkin F. Suarez-Huerta N. Robaye B. Peetermans J. Libert F. Dumont J.E. Maenhaut C. Eur. J. Biochem. 1997; 248: 660-668Crossref PubMed Scopus (52) Google Scholar). Quantitative real time-PCR experiments showed that trb-1 mRNA was expressed in most human tissues with the highest levels in skeletal muscle, thyroid gland, pancreas, peripheral blood leukocytes, and bone marrow (Supplementary Fig. 1). trb-2 levels were highest in peripheral blood leukocytes, and trb-3 levels were highest in pancreas peripheral blood leukocytes and bone marrow; in addition HeLa cells were found to express trbs 1–3. (Supplementary Fig. 1). Trbs Regulate AP-1—When overexpressed, Htrb-1 repressed basal activity of the NFκB and AP-1-regulated IL-8 promoter in HeLa cells in culture but not its activation by IL-1 and tumor necrosis factor α (Fig. 1A). The NFκB sites mediate cytokine activation, whereas the AP-1 sites control basal activity (22Mukaida N. Okamoto S. Ishikawa Y. Matsushima K. J. Leukocyte Biol. 1994; 56: 554-558Crossref PubMed Scopus (410) Google Scholar). Our results therefore suggest that Trb-1 acts selectively on pathways leading to AP-1. Consistent with this, testing specific NFκB or AP-1 reporters activated by MEKK-1 co-transfection showed that Htrb-1 inhibited AP-1 activity but not NFκB (Fig. 1, B and C) nor a signal transducers and activators of transcription-responsive LHRE promoter (Fig. 1D). Specificity was also analyzed by testing a panel of human cytokines in the presence or absence of Htrb-3; AP-1 activation was inhibited (Fig. 1H), whereas NFκB activation was not (Supplementary Table I). Thus, whether activated by overexpression of upstream components or by physiological agonists, pathways leading to AP-1 activation were specifically inhibited by Trb overexpression. We have shown above that up-regulation of endogenous Trb-1 and ectopic overexpression of Trb-1 and Trb-3 inhibited AP-1 activity in HeLa cells. Next we examined the effects of suppressing endogenous Trb expression. An antisense construct encoding the htrb-3 5′-untranslated region and N-terminal domain was co-transfected with AP-1 or NFκB reporters using either MEKK-1 or NFκB inducing kinase as activators (Fig. 1, E–F). Trb-3 mRNA expression detected by quantitative real time-PCR using primers spanning the central Trb domain was reduced by >70% (Fig. 1G), whereas Trb-1 levels were only slightly altered by transfecting a high dose of AS-Trb-3 construct, suggesting a Trb-3-specific action. As observed with overexpression, AP-1 but not NF-κB activity was inhibited (Fig. 1, E and F). Trbs Regulate ERKs—Because Trbs are mitogen-induced and regulate String/cdc25, we examined the effect of Trb levels on the control of the Ras/MEK/ERK module. V12Ras-driven AP-1 activation was blocked by overexpression of both Trb-1 and Trb-3 (Fig. 2A). Also, HeLa cells were stimulated with PMA, and phospho-ERK levels were determined by Western blotting; basal levels were increased by Htrb-3, and both the extent and rate of phosphorylation were enhanced by Htrb-1 or Htrb-3 (Fig. 2B), correlating with enhanced ERK activity measured in an in vitro kinase assay (Fig. 2D). Although expression levels of Trb-1 and Trb-3 were comparable (see Fig. 4), ERK activation was enhanced only using low doses of Trb-3 (Fig. 2C), and ERK potentiation was enhanced in the presence of all doses of Trb-1 within the range tested (Fig. 2C), suggesting that the system is more sensitive toward Trb-3 levels. Comparison between constitutive (transfection, Fig. 2A) and transient (PMA treatment, Fig. 2, B–D) stimuli showed that Trbs are capable of either upor down-regulating MAPK activity.Fig. 4Trbs interact with MAPKK specifically.A, titration of Myc-htrb and FLAG-MAPKK protein levels after co-transfection. The side labels show developing antibody, MKK, and Trb constructs, whereas below the bands are micrograms of plasmids co-transfected. B, to define the specific interacting partners, the indicated tagged proteins were immunoprecipitated with the indicated antibody and immunoblotted with anti-Myc. Lanes 1, 3, 5, 7, 9, and 11 are negative controls for interactions between the FLAG-tagged protein and cellular Myc. Lanes 2, 4, 6, 8, 10, and 12 are experimental lanes for interactions between the FLAG-tagged proteins and Htrb-1 and Htrb-3 with the FLAG-tagged are indicated by the results from the cell that the is in both tagged proteins were immunoprecipitated with either or and immunoblotted with both antibodies Lanes and are negative controls for interactions between the transfected protein and the Lanes 2 and 4 are the experimental lanes for interactions between the and FLAG-tagged Htrb-3 or and MAPKK with of htrb-1 inhibited MEKK-1 AP-1 but not NFκB activity (Fig. 1, B and a effect was an AP-1 reporter was stimulated by (Fig. characterize the site of we tested whether the dose of (Fig. or MEKK-1 (Fig. the effect of This was not the suggesting that htrb-3 controls a of MEKK-1 and activation of kinases to phosphorylation of transcription including c-Jun and Western showed that both the and the extent by of phosphorylation were altered by Trb-3 (Fig. the of of trbs in the pathway is at or of and at or upstream of p38 MAPK was not activated by in However, overexpression of Trb proteins inhibited basal p38 activity as detected in an in vitro kinase assay (Fig. These data suggest that Trbs at the level of and hence control MAPK the of we used transfection and experiments with Trbs and FLAG-tagged and MAPKK We found that with both Trb-1 and specifically with and specifically with Trb-1 (Fig. interactions were detected between Trb and or p38 not These were in 2 interactions were detected between both Trbs and between Trb-3 and but not between Trb and not These data are with the results of the assays 2 and suggesting that trbs control MAPK activation by to (Fig. for all (Fig. showed that Htrb-3 levels were increased by co-transfection (Fig. and but not which not bind to Htrb-1 levels were enhanced by but not which not bind to interactions In effect was on MAPKK as by Fig. These are with the and 2 and suggest that Trb protein levels may be regulated by Trbs Regulate the of Trbs interact with more than one MAPKK and to have effects on ERK, JNK, and p38 and 3, and activation, we examined whether Trbs regulate MAPK activation. Htrb-3 with both and and in HeLa we examined Htrb-3 using MAPK reporter assays to between ERK, JNK, and low Htrb-3 enhanced and ERK activation a more effect on but inhibited p38 (Fig. doses the activation of all three pathways was inhibited relative to to be a different for each pathway (Fig. suggesting that may regulate relative activation of the three classes of MAPK. We show that Trbs for kinase pathway function and are at high levels. mechanisms can for Trbs be both and of which has been shown to MAPK signaling M. K. T. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, J.E. 2000; Google Scholar, A. J. S. A. 2000; PubMed Scopus Google Scholar, J. A.J. J. M. Davis R.J. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). Our data show that not only bind Trbs but also to Further, Trb proteins have been reported to be rapidly mitogen and to have (9Wilkin F. Suarez-Huerta N. Robaye B. Peetermans J. Libert F. Dumont J.E. Maenhaut C. Eur. J. Biochem. 1997; 248: 660-668Crossref PubMed Scopus (52) Google Scholar, 10Wilkin F. Savonet V. Radulescu A. Petermans J. Dumont J.E. Maenhaut C. J. Biol. Chem. 1996; 271: 28451-28457Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), and we have found that Trb are also rapidly by and have data suggest that interactions are and regulated by In addition, MAPK cascades have been to a leading to J.E. 2000; Google Scholar). using that with in Trb proteins either as activators or as of MAPK depending on the of Trb to MAPKK levels in the K. Dower and E. Kiss-Toth, unpublished data. have been by and for the NFκB F. R. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (61) Google Scholar, A. A. D. Science. 2002; PubMed Scopus Google Scholar) and the cell control A. A. B. B. S. A. 2000; PubMed Scopus Google Scholar). We also that are capable of more than the dose we for the observed in cells from and in A. A. D. 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Yoshioka for the and We also J. for the V12 Ras and S. for with

Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.

Our goal is to construct an improved Escherichia coli to serve both as a better model organism and as a more useful technological tool for genome science. We developed techniques for precise genomic surgery and applied them to deleting the largest K-islands of E. coli, identified by comparative genomics as recent horizontal acquisitions to the genome. They are loaded with cryptic prophages, transposons, damaged genes, and genes of unknown function. Our method leaves no scars or markers behind and can be applied sequentially. Twelve K-islands were successfully deleted, resulting in an 8.1% reduced genome size, a 9.3% reduction of gene count, and elimination of 24 of the 44 transposable elements of E. coli. These are particularly detrimental because they can mutagenize the genome or transpose into clones being propagated for sequencing, as happened in 18 places of the draft human genome sequence. We found no change in the growth rate on minimal medium, confirming the nonessential nature of these islands. This demonstration of feasibility opens the way for constructing a maximally reduced strain, which will provide a clean background for functional genomics studies, a more efficient background for use in biotechnology applications, and a unique tool for studies of genome stability and evolution.

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17(-) deletion mutant compared with the isogenic wild-type hsp17(+) revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a "membrane stabilizing factor" as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

Understanding how evolution of antimicrobial resistance increases resistance to other drugs is a challenge of profound importance. By combining experimental evolution and genome sequencing of 63 laboratory-evolved lines, we charted a map of cross-resistance interactions between antibiotics in Escherichia coli, and explored the driving evolutionary principles. Here, we show that (1) convergent molecular evolution is prevalent across antibiotic treatments, (2) resistance conferring mutations simultaneously enhance sensitivity to many other drugs and (3) 27% of the accumulated mutations generate proteins with compromised activities, suggesting that antibiotic adaptation can partly be achieved without gain of novel function. By using knowledge on antibiotic properties, we examined the determinants of cross-resistance and identified chemogenomic profile similarity between antibiotics as the strongest predictor. In contrast, cross-resistance between two antibiotics is independent of whether they show synergistic effects in combination. These results have important implications on the development of novel antimicrobial strategies.