Molecular glue degraders: exciting opportunities for novel drug discovery.

INTRODUCTION: Molecular Glue Degraders (MGDs) is a concept that refers to a class of compounds that facilitate the interaction between two proteins or molecules within a cell. These compounds act as bridge that enhances specific Protein-Protein Interactions (PPIs). Over the past decade, this technology has gained attention as a potential strategy to target proteins that were traditionally considered undruggable using small molecules. AREAS COVERED: This review presents the concept of cellular homeostasis and the balance between protein synthesis and protein degradation. The concept of protein degradation is concerned with molecular glues, which form part of the broader field of Targeted Protein Degradation (TPD). Next, pharmacochemical strategies for the rational design of MGDs are detailed and illustrated by examples of Ligand-Based (LBDD), Structure-Based (SBDD) and Fragment-Based Drug Design (FBDD). EXPERT OPINION: Expanding the scope of what can be effectively targeted in the development of treatments for diseases that are incurable or resistant to conventional therapies offers new therapeutic options. The treatment of microbial infections and neurodegenerative diseases is a major societal challenge, and the discovery of MGDs appears to be a promising avenue. Combining different approaches to discover and exploit a variety of innovative therapeutic agents will create opportunities to treat diseases that are still incurable.

Promoting vaccination in maternity wards ─ motivational interview technique reduces hesitancy and enhances intention to vaccinate, results from a multicentre non-controlled pre- and post-intervention RCT-nested study, Quebec, March 2014 to February 2015.

BackgroundMany countries are grappling with growing numbers of parents who delay or refuse recommended vaccinations for their children. This has created a need for strategies to address vaccine hesitancy (VH) and better support parental decision-making regarding vaccination.AimTo assess vaccination intention (VI) and VH among parents who received an individual motivational-interview (MI) based intervention on infant immunisation during post-partum stay at a maternity ward between March 2014 and February 2015.MethodsThis non-controlled pre-/post-intervention study was conducted using the results from parents enrolled in the intervention arm of the PromoVaQ randomised control trial (RCT), which was conducted in four maternity wards across the Province of Quebec. Participants (n = 1,223) completed pre- and post-intervention questionnaires on VI and VH using Opel's score. Pre-/post-intervention measures were compared using McNemar's test for categorical variables and Wilcoxon signed-rank test for continuous variables.ResultsPre-intervention: overall VI was 78% and significantly differed across maternity wards (74%, 77%, 84%, 79%, p = 0.02). Post-intervention: VI rose significantly across maternity wards (89%, 85%, 95%, 93%) and the overall increase in VI was 12% (78% vs 90%, p < 0.0001). VH corroborated these observations, pre- vs post-intervention, for each maternity ward (28% vs 16%, 29% vs 21%, 27% vs 17%, 24% vs 13%). Overall, VH was curbed post-intervention by 40% (27% vs 16%; p < 0.0001).ConclusionsCompared with pre-intervention status, participants who received the MI-based intervention on immunisation displayed lower hesitancy and greater intention to vaccinate their infant at 2 months of age.

Promoting vaccination in the province of Québec: the PromoVaQ randomized controlled trial protocol.

BACKGROUND: Vaccination has a huge public health impact. Maintaining vaccine coverage is key to avoid the devastating consequences of resurgence. In the Province of Québec, vaccine coverage in young children are sub-optimal, mostly due to ambivalence toward vaccine safety and efficacy. We previously conducted a regional study in the Québec's Eastern Townships region, the PromoVac Study, to test a new educational intervention, based on motivational interviewing techniques, aimed at promoting infant vaccination. This first study evidenced that the intervention led to a marked increase in mothers' intention to vaccinate, and vaccine coverage in their infants. The current study protocol aims at scaling up these results at a provincial level using a randomized controlled trial design. METHODS: This pragmatic, randomized, controlled, parallel-group clinical trial will compare the effectiveness of the motivational interviewing to an educational intervention, including the distribution of an information flyer as standard of care on vaccination coverage in four maternity wards across the Province of Québec (PromovaQ). Adult mothers of children born in participating maternity wards were recruited between March 2014 and February 2015. Vaccination coverage will be assessed at 3-years of age, thus the trial is expected to be completed in March 2019. Statistical analyses will be conducted under the intention-to-treat principle. Vaccine coverage will be analyzed using Chi-squared distribution testing and logistic regression to identify determinant factors. Secondary outcomes will include vaccine hesitation and intention scores, mother's knowledge, attitudes and beliefs about immunization, and psychosocial determinants of intention to vaccinate. DISCUSSION: In the case results of this Provincial RCT be confirmed, serious consideration should then be given by Ministry of Health authorities to the possible implementation of MI-based strategies across provincial maternity wards. To ensure adequate input and secure implementation, study design and results will be reviewed with relevant stakeholders, including the children's families, and provincial and regional decision-makers. Results will be adapted and shared with all stakeholders. TRIAL REGISTRATION: ClinicalTrials.gov NCT02666872 (Retrospectively registered as January 28, 2016).

Overview of knowledge, attitudes, beliefs, vaccine hesitancy and vaccine acceptance among mothers of infants in Quebec, Canada.

BACKGROUND: Vaccine hesitancy (VH) is a growing problem. The first step in addressing VH is to have an understanding of who are the hesitant individuals and what are their specific concerns. The aim of this survey was to assess mothers' level of vaccine hesitancy and vaccination knowledge, attitudes, and beliefs. METHODS: Mothers of newly-born infants in four maternity wards in Quebec (Canada) completed a self-administered questionnaire. The questionnaire included items to assess VH and intention to vaccinate. VH scores were calculated using the Parents Attitudes about Childhood Vaccines (PACV) survey. Multivariate logistic regression was performed to determine variables associated with intention to vaccinate (OR; 95% CI). RESULTS: Overall, 2645 questionnaires were included in this analysis and 77.5% of respondents certainly intended to vaccinate their infant at 2 months of age. Based on the PACV 100-point scale, 56.4% of mothers had a 0 to ˂30 score (low level of VH); 28.6% had a 30 to ˂50 and 15.0% had a score of 50 and higher (high level of VH).The main determinants of mothers' intention to vaccinate were the perceived importance of vaccinating infants at 2 months of age (OR = 9.2; 5.9-14.5) and a low score of VH (OR = 7.4; 5.3-10.3). DISCUSSION: Although the majority of mothers held positive attitudes toward vaccination, a large proportion were moderately or highly vaccine hesitant. Mothers' level of VH was strongly associated with their intention to vaccinate their infants, showing the potential detrimental impact of VH on vaccine uptake rates and the importance of addressing this phenomenon.

Impact of a vaccination promotion intervention using motivational interview techniques on long-term vaccine coverage: the PromoVac strategy.

BACKGROUND: Delayed vaccinations at 2, 4, and 6 months are associated with a higher probability of delayed age-appropriate vaccination during childhood. This study aimed to assess the effectiveness of an information session on immunization during infancy. METHODS: An individual educational information session with motivational interview techniques for immunization of infants was conducted (experimental group) or not conducted (control group) during postpartum stay in a quasi-experimental cohort study. Immunization data were collected from the Eastern Townships Public Health registry at 3, 5, 7, 13, 19, and 24 months of age. Logistic regressions with repeated measures were performed to assess the intervention's impact. Relative risks (RR) were estimated. A multivariate model was obtained adjusted for confounding factors. RESULTS: The experimental and control groups included 1140 and 1249 families, respectively. In per protocol analysis, a significant increase in VC of 3.2, 4.9, 7.3, 6.7, 10.6, and 5.1% was observed at 3, 5, 7, 13, 19, and 24 months. Children from experimental group had 9% more chance at a complete vaccination status between 3 and 24 months compared to children from control group (RR (95% CI): 1.09 (1.05-1.13), p < .001). Children with complete vaccination status at 3 months were more likely to have a complete vaccination status at 24 months (82.3 vs. 48.1%, RR (95% CI): 2.72 (2.28-3.24), p < .001). After adjustment, the estimated RR of the intervention's impact was 1.05 (1.02-1.07), p < .001. CONCLUSIONS: An educational information session about immunization based on motivational interview techniques conducted during postpartum hospitalization could improve immunization during infancy.

A postpartum vaccination promotion intervention using motivational interviewing techniques improves short-term vaccine coverage: PromoVac study.

BACKGROUND: Due to the increasing number of vaccine-hesitant parents, new effective immunization promotion strategies need to be developed to improve the vaccine coverage (VC) of infants. This study aimed to assess the impact of an educational strategy of vaccination promotion based on motivational interviewing (MI) techniques targeting parents and delivered at the maternity ward, for the VC of infants at 3, 5, and 7 months of age. METHODS: An individual educational information session, administered using MI techniques, regarding immunization of infants aged 2, 4, and 6 months was (experimental group) or was not (control group) proposed to parents during the postpartum stay at the maternity ward. Immunization data were obtained through the Eastern Townships Public Health registry for infants at 3, 5, and 7 months of age. Absolute VC increases at 3, 5, and 7 months in the experimental group were calculated and the relative risks with the respective 95% confidence intervals were computed using univariate logistic regression with the generalized estimating equations (GEE) procedure. Multivariate regression using GEE was used to adjust for confounding variables. RESULTS: In the experimental and control groups, 1140 and 1249 newborns were included, respectively. A significant increase in VC of 3.2, 4.9, and 7.3% was observed at 3, 5, and 7 months of age (P < 0.05), respectively. The adjusted relative risk of the intervention's impact on vaccination status at 7 months of age was 1.08 (95% confidence interval: 1.03-1.14) (P = 0.002). CONCLUSIONS: An educational strategy using MI techniques delivered at the maternity ward may be effective in increasing VC of infants at ages 3, 5, and 7 months. MI could be an effective tool to overcome vaccine hesitancy.

Impact of rotavirus vaccine on premature infants.

Infants born preterm are at a higher risk of complications and hospitalization in cases of rotavirus diarrhea than children born at term. We evaluated the impact of a rotavirus vaccination campaign (May 2007 to May 2010) on hospitalizations for rotavirus gastroenteritis in a population of children under 3 years old born prematurely (before 37 weeks of gestation) in the Brest University Hospital birth zone. Active surveillance from 2002 to 2006 and a prospective collection of hospitalizations for rotavirus diarrhea were initiated in the pediatric units of Brest University Hospital until May 2010. Numbers of hospitalizations for rotavirus diarrhea among the population of children born prematurely, before and after the start of the vaccination program, were compared using a Poisson regression model controlling for epidemic-to-epidemic variation. A total of 217 premature infants were vaccinated from 2007 to 2010. Vaccine coverage for a complete course of three doses was 41.9%. The vaccine safety in premature infants was similar to that in term infants. The vaccination program led to a division by a factor of 2.6 (95% confidence interval [CI], 1.3 to 5.2) in the number of hospitalizations for rotavirus diarrhea during the first two epidemic seasons following vaccine introduction and by a factor of 11 (95% CI, 3.5 to 34.8) during the third season. We observed significant effectiveness of the pentavalent rotavirus vaccine on the number of hospitalizations in a population of prematurely born infants younger than 3 years of age. A multicenter national study would provide better assessment of this impact. (This study [Impact of Systematic Infants Vaccination Against Rotavirus on Gastroenteritis Hospitalization: a Prospective Study in Brest District, France (IVANHOE)] has been registered at ClinicalTrials.gov under registration no. NCT00740935.).

Maternal knowledge, attitudes and beliefs regarding gastroenteritis and rotavirus vaccine before implementing vaccination program: which key messages in light of a new immunization program?

In July 2010, the National Advisory Committee on Immunization (NACI) recommended the systematic administration of rotavirus vaccines for all infants in Canada. According to the Erickson and De Wals framework, multiple factors need to be evaluated before implementing such a decision, including the study of the acceptability of this vaccine by the general population. A cross-sectional survey was conducted from February 10 to February 18, 2011, at the Sherbrooke University Hospital Center in the province of Quebec. A questionnaire, based upon the Health Belief Model (HBM) and theoretical planned action, was self-administered to pregnant or early post-partum women. The variables collected included socio-demographic data, past experience with gastroenteritis, cues to vaccination and HBM dimensions. The associations between questionnaire variables and vaccination intention were assessed using univariate and multivariate analyses. Of the 343 respondents, only 29% had already heard about rotavirus vaccination and among these, the intention of vaccination was 74%. In multivariate analysis, having a perception of infant vulnerability to gastroenteritis (OR=2.3, 95% CI 1.3-4.0) and having no other child at home (OR=2.3, 95% CI 1.3-4.2) were factors positively associated with a higher intention of vaccination, contrary to having already heard about the rotavirus vaccine in the media (OR=0.5, 95% CI 0.2-0.9). The three cues independently associated with intention of vaccination were the reimbursement of the vaccine (OR=3.0, 95% CI 1.6-5.7), its recommendation by a doctor (OR=21.2, 95% CI 5.8-75.9) and its protection against the most severe forms of gastroenteritis (OR=4.4, 95% CI 1.4-13.6). To improve the success of this new vaccination program, several key messages should be integrated in the information made available to the general population: (1) rotavirus gastroenteritis is a mandatory infection for every child <5 years; (2) the vaccine is reimbursed and included in the provincial vaccination program; and (3) the vaccine protects against the worst forms of gastroenteritis. Finally, support should be offered to physicians as they play a key role in public acceptance of new vaccines.

Soluble and filamentous proteins in Arabidopsis sieve elements.

Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.

Impact of rotavirus vaccination on hospitalizations for rotavirus diarrhea: the IVANHOE study.

The aim of the IVANHOE study was to determine the real-world impact of the rotavirus vaccine, controlling for epidemic-to-epidemic variation in disease burden. A population-based prospective cohort study was conducted in Brest City and 7 suburban districts (CUB area), North-western Brittany, France (210,000 inhabitants; 5500 births per year). The vaccination program started in May 2007 for a 2-year period for all infants born in the Brest birth zone through pediatricians, public outpatient clinics and general practitioners. To determine vaccine impact we monitored trends in hospitalizations for rotavirus-specific diarrhea using an active hospital-based surveillance system initiated 5 years before vaccine introduction. The number of hospitalizations for rotavirus-specific diarrhea during the 2008/2009 epidemic in infants less than 2 years of age whose parents lived within the CUB area was modelled as a function of (1) the number of hospitalizations in infants 2-5 years of age to control for epidemic-to-epidemic variation and (2) vaccine introduction. A total of 4684 infants received at least one dose. Of these, 2635 lived within the CUB area. Vaccine coverage for a complete schedule in the CUB area was 47.1%. Poisson modelling revealed a reduction by a factor of 2.04 (1.56-2.66) in the number of hospitalizations during the last epidemic season (2008/2009), the number of observed cases being equal to 30, against an expected number of 61. Relative risk reduction for hospitalizations for rotavirus diarrhea was 98% (95% CI: 83-100%). We observed a noticeable impact of vaccination on rotavirus diarrhea hospitalizations within 2 years of vaccine introduction integrating for the first time rotavirus epidemics variation. The trial is registered with ClinicalTrials.gov, number, NCT00740935.

The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting.

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using (15)N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.

Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession.

Adaptation to steady-state low-nutrient availability was investigated by comparing the Wassileskija (WS) accession of Arabidopsis thaliana grown on 2 or 10 mM nitrate. Low nitrogen conditions led to a limited rosette biomass and seed yield. The latter was mainly due to reduced seed number, while seed weight was less affected. However, harvest index was lower in high nitrate compared with limited nitrate conditions. Under nitrogen-limiting conditions, nitrate reductase activity was decreased while glutamine synthetase activity was increased due to a higher accumulation of the cytosolic enzyme. The level of nitrogen remobilization to the seeds was higher under low nitrogen, and the vegetative parts of the plants remaining after seed production stored very low residual nitrogen. Through promoting nitrogen remobilization and recycling pathways, nitrogen limitation modified plant and seed compositions. Rosette leaves contained more sugars and less free amino acids when grown under nitrogen-limiting conditions. Compared with high nitrogen, the levels of proline, asparagine and glutamine were decreased. The seed amino acid composition reflected that of the rosette leaves, thus suggesting that phloem loading for seed filling was poorly selective. The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.

Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition.

Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of (15)N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early- and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.

NAD-dependent isocitrate dehydrogenase mutants of Arabidopsis suggest the enzyme is not limiting for nitrogen assimilation.

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.

Genetic variation suggests interaction between cold acclimation and metabolic regulation of leaf senescence.

The extent to which leaf senescence is induced by nitrogen deficiency or by sugar accumulation varies between natural accessions of Arabidopsis (Arabidopsis thaliana). Analysis of senescence in plants of the Bay-0 x Shahdara recombinant inbred line (RIL) population revealed a large variation in developmental senescence of the whole leaf rosette, which was in agreement with the extent to which glucose (Glc) induced senescence in the different lines. To determine the regulatory basis of genetic differences in the Glc response, we investigated changes in gene expression using Complete Arabidopsis Transcriptome MicroArray (CATMA) analysis. Genes whose regulation did not depend on the genetic background, as well as genes whose regulation was specific to individual RILs, were identified. In RIL 310, a line that does not show the typical senescence response to Glc, stress response genes, especially those responding to cold stress, were induced by Glc. We therefore tested whether cold acclimation delays senescence by reducing sugar sensitivity. In cold-acclimated plants, leaf senescence was severely delayed and Glc did not induce the typical senescence response. Together, our results suggest that cold acclimation extends rosette longevity by affecting metabolic regulation of senescence, thereby allowing vernalization-dependent plants to survive the winter period. The role of functional chloroplasts and of nitrogen and phosphate availability in this regulation is discussed.

Expression analysis of Arabidopsis thaliana NAD-dependent isocitrate dehydrogenase genes shows the presence of a functional subunit that is mainly expressed in the pollen and absent from vegetative organs.

NAD-dependent isocitrate dehydrogenase (IDH) is a Krebs cycle enzyme situated in mitochondria. In Arabidopsis thaliana, five genes encode functional IDH subunits that can be classed into two groups based on gene structure and subunit amino acid sequence. Arabidopsis contains two 'catalytic' and three 'regulatory' subunits according to their homology with yeast IDH. To date, an active IDH is believed to be heteromeric, containing at least one of each subunit type. This was verified in Arabidopsis by the complementation of yeast IDH mutants with the different Arabidopsis IDH-encoding cDNAs. Indeed, a single 'catalytic' and 'regulatory' subunit was sufficient to restore acetate growth of the yeast IDH double mutant. To gain information on possible IDH subunit interactions in planta, Arabidopsis IDH gene expression was analysed by Northern blot, PCR on cDNA libraries, in silico and in 'promoter'-reporter gene transgenic plants. Four of the IDH genes were expressed in all plant organs tested, while one gene (At4g35650) was not expressed in vegetative organs but was mainly expressed in the pollen. In leaves, the IDH genes were highly expressed in the veins, and to a lesser extent in mesophyll cells. The data are discussed with respect to IDH in other plant species.

AKINbeta3, a plant specific SnRK1 protein, is lacking domains present in yeast and mammals non-catalytic beta-subunits.

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic beta-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINbeta3, a novel protein related to AKINbeta subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (beta-GBD) identified in the mammalian AMPKbeta1. In spite of its unusual features, AKINbeta3 complements the yeast sip1Deltasip2Deltagal83Delta mutant. Moreover, interactions between AKINbeta3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINbeta3 is a beta-type subunit. A search for beta-type subunits revealed the existence of beta3-type proteins in other plant species. Furthermore, we suggest that the AKINbeta3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINbeta3-type subunits, involved in several functions among which some could be plant specific.