The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC.

A repressor of the transition to flowering in Arabidopsis is the MADS box protein FLOWERING LOCUS C (FLC). FCA, an RNA-binding protein, and FY, a homolog of the yeast RNA 3' processing factor Pfs2p, downregulate FLC expression and therefore promote flowering. FCA/FY physically interact and alter polyadenylation/3' processing to negatively autoregulate FCA. Here, we show that FCA requires FLOWERING LOCUS D (FLD), a homolog of the human lysine-specific demethylase 1 (LSD1) for FLC downregulation. FCA also partially depends on DICER-LIKE 3, involved in chromatin silencing. fca mutations increased levels of unspliced sense FLC transcript, altered processing of antisense FLC transcripts, and increased H3K4 dimethylation in the central region of FLC. These data support a close association of FCA and FLD in mediating H3K4 demethylation and thus transcriptional silencing of FLC and reveal roles for antisense RNA processing and DCL3 function in this regulation.

Calcium binding proteins in the liver fluke, <em>Fasciola hepatica</em>

The common liver fluke, Fasciola hepatica, is a parasite of mammals. In the western world its effects are largely felt on agriculture where infection of cows, sheep and other farm animals is estimated to cause millions of dollars ofif financial losses. In the developing world, the problem is even more serious with an estimated 7 million infected people and many millions more at risk of infection. Calcium signalling is of key importance in all eukaryotic species and recent discoveries of novel types of calcium binding proteins in liver flukes (and related trematodes) suggest that there may be calcium signalling processes which are unique to this group of organisms. If so, these pathways may provide potential targets for the design of novel anthelmintic drugs. Here, we review three main groups of F. hepatica calcium binding proteins: the FH8 family, the calmodulin family (FhCaM1, FhCaM2 and FhCaM3) and the EF-hand/dynein light chain family (FH22, FhCaBP3, FhCaBP4). Considerable information has been gathered on the sequences, predicted structures and biochemical properties of these molecules. The challenge now is to understand their functions in the organism.

The identification of p130cas-binding proteins and their role in cellular transformation

Adaptor proteins play an important role in signal transduction by regulating the establishment and maintenance of functionally important protein complexes. A recently described member of this group of proteins is p130cas (CAS), which contains numerous sequence motifs predicted to be involved in mediating protein-protein interactions. We propose that adaptor molecules like CAS may help determine the response of a cell to a particular signal by interacting with specific subsets of cellular proteins. To test this hypothesis, we have identified potential binding partners of CAS that may play a rote in cellular transformation by the oncoproteins v-SRC and/or v-CRK. We show that individual domains of CAS associate with specific subsets of proteins in vitro, and that many of these interactions are dependent on the state of tyrosine-phosphorylation of CAS. Sequences necessary for interacting with the focal adhesion kinase pp125FAK (FAK), v-SRC and v-CRK have been mapped to distinct regions of CAS. In addition, the identification of a number of putative CAS-binding partners that are present in crk-transformed cell extracts but undetectable in normal and src-transformed cell extracts supports a model in which unique protein complexes are formed in response to different signals.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Peptidoglycan assembly machines: The Staphylococcus aureus Penicillin-Binding Proteins

Dissertation presented to obtain the Ph.D degree in Biology

Regulatory RNA binding molecules : implication for diabetes pathogenesis

Le glucose est notre principale source d'��nergie. Apr��s un repas, le taux de glucose dans le sang (glyc��mie) augmente, ce qui entraine la s��cr��tion d'insuline. L'insuline est une hormone synth��tis��e au niveau du pancr��as par des cellules dites b��ta. Elle agit sur diff��rents organes tels que les muscles, le foie ou le tissu adipeux, induisant ainsi le stockage du glucose en vue d'une utilisation future.��Le diab��te est une maladie caract��ris��e par un taux ��lev�� de glucose dans le sang (hyperglyc��mie), r��sultant d'une incapacit�� de notre corps �� utiliser ou �� produire suffisamment d'insuline. A long terme, cette hyperglyc��mie entra��ne une d��t��rioration du syst��me cardio-vasculaire ainsi que de nombreuses complications. On distingue principalement deux type de diab��te : le diab��te de type 1 et le diab��te de type 2, le plus fr��quent (environ 90% des cas). Bien que ces deux maladies diff��rent sur beaucoup de points, elles partagent quelques similitudes. D'une part, on d��c��le une diminution de la quantit�� de cellules b��ta. Cette diminution est cependant partielle dans le cas d'un diab��te de type 2, et totale dans celui d'un diab��te de type 1. D'autre part, la pr��sence dans la circulation de m��diateurs de l'inflammation nomm��s cytokines est d��cel��e aussi bien chez les patients de type 1 que de type 2. Les cytokines sont s��cr��t��es lors d'une inflammation. Elles servent de moyen de communication entre les diff��rents acteurs de l'inflammation et ont pour certaines un effet n��faste sur la survie des cellules b��ta.��L'objectif principal de ma th��se a ��t�� d'��tudier en d��tail l'effet de petites mol��cules r��gulatrices de l'expression g��nique, appel��es microARNs. Bas�� sur le fait que de nombreuses publications ont d��montr�� que les microARNs ��taient impliqu��s dans diff��rentes maladies telles que le cancer, j'ai ��mis l'hypoth��se qu'ils pouvaient ��galement jouer un r��le important dans le d��veloppement du diab��te.��Nous avons commenc�� par mettre des cellules b��ta en culture en pr��sence de cytokines, imitant ainsi un environnement inflammatoire. Nous avons pu de ce fait identifier les microARNs dont les niveaux d'expression ��taient modifi��s. A l'aide de m��thodes biochimiques, nous avons ensuite observ�� que la modulation de certains microARNs par les cytokines avaient des effets n��fastes sur la cellule b��ta : sur sa production et sa s��cr��tion d'insuline, ainsi que sur sa mort (apoptose). Nous avons en cons��quence pu d��montrer que ces petites mol��cules avaient un r��le important �� jouer dans le dysfonctionnement des cellules b��ta induit par les cytokines, aboutissant au d��veloppement du diab��te.��-��La cellule b��ta pancr��atique est une cellule endocrine pr��sente dans les ��lots de Langerhans, dans le pancr��as. L'insuline, une hormone s��cr��t��e par ces cellules, joue un r��le essentiel dans la r��gulation de la glyc��mie. Le diab��te se d��veloppe si le taux d'insuline rel��ch�� par les cellules b��ta n'est pas suffisant pour couvrir les besoins m��taboliques corporels. Le diab��te de type 1, qui repr��sente environ 5 �� 10% des cas, est une maladie auto-immune qui se caract��rise par une r��action inflammatoire d��clench��e par notre syst��me immunitaire envers les cellules b��ta. La cons��quence de cette attaque est une disparition progressive des cellules b��ta. Le diab��te de type 2 est, quant �� lui, largement plus r��pandu puisqu'il repr��sente environ 90% des cas. Des facteurs �� la fois g��n��tiques et environnementaux sont responsables d'une diminution de la sensibilit�� des tissus m��tabolisant l'insuline, ainsi que d'une r��duction de la s��cr��tion de l'insuline par les cellules b��ta, ce qui a pour cons��quence le d��veloppement de la maladie. Malgr�� les diff��rences entre ces deux types de diab��te, ils ont pour points communs la pr��sence d'infiltrat immunitaire et la diminution de l'��tat fonctionnel des cellules b��ta.��Une meilleure compr��hension des m��canismes aboutissant �� l'alt��ration de la cellule b��ta est primordiale, avant de pouvoir d��velopper de nouvelles strat��gies th��rapeutiques capables de gu��rir cette maladie. Durant ma th��se, j'ai donc ��tudi�� l'implication de petites mol��cules d'ARN, r��gulatrices de l'expression g��nique, appel��es microARNs, dans les conditions physiopathologiques qui aboutissent au d��veloppement du diab��te. J'ai d��but�� mon ��tude par l'identification de microARNs dont le niveau d'expression ��tait modifi�� lorsque les cellules b��ta ��taient expos��es �� des conditions favorisant �� la fois le d��veloppement du diab��te de type 1 (cytokines) et celui du diab��te de type 2 (palmitate). Nous avons d��couvert qu'une modification de l'expression des miR-21, -34a et -146a ��tait commune aux deux traitements. Ces changements d'expressions ont ��galement ��t�� confirm��s dans deux mod��les animaux : les souris NOD qui d��veloppent un diab��te s'apparentant au diab��te de type 1 et les souris db/db qui d��veloppent plut��t un diab��te de type 2. Puis, �� l'aide de puces �� ADN, nous avons compar�� l'expression de microARNs chez des souris NOD pr��-diab��tiques. Nous avons alors retrouv�� des changements au niveau de l'expression des m��mes microARNs mais ��galement au niveau d'une famille de microARNs : les miR-29a, -29b et -29c. De mani��re artificielle, nous avons ensuite surexprim�� ou inhib�� en conditions physiopathologiques l'expression de tous ces microARNs et nous nous sommes int��ress��s �� l'impact d'un tel changement sur diff��rentes fonctions de la cellule b��ta comme la synth��se et la s��cr��tion d'insulin�� ainsi que leur survie. Nous avons ainsi pu d��montrer que les miR-21, -34a, -29a, -29b, -29c avaient un effet d��l��t��re sur la s��cr��tion d'insuline et que la surexpression de tous ces microARNs (except�� le miR-21) favorisait la mort. Finalement, nous avons d��montr�� que la plupart de ces microARNs ��taient impliqu��s dans la r��gulation d'importantes voies de signalisation responsables de l'apoptose des cellules b��ta telles que les voies de NFKB, BCL2 ou encore JNK.��Par cons��quent, nos r��sultats d��montrent que les microARNs ont un r��le important �� jouer dans le dysfonctionnement des cellules b��ta lors de la mise en place du diab��te.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as ���ladybug taint���. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.

Purification and identification of specific RNA-binding protein that binds to the 3'UTR region of cytochrome P450aromatase mRNA in bovine granulosa cells

M��moire num��ris�� par la Division de la gestion de documents et des archives de l'Universit�� de Montr��al

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine

Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered.

Small guanine nucleotide-binding proteins and myocardial hypertrophy.

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.