Quantitative tandem affinity purification, an effective tool to investigate protein complex composition in plant hormone signaling : strigolactones in the spotlight

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones

Unraveling the MAX2 protein network in Arabidopsis thaliana : identification of the protein phosphatase PAPP5 as a novel MAX2 interactor

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway

Paediatric trauma in Flanders (Belgium): are we forgetting the pain?

status: accepte

Paeditric trauma in Flanders (Belgium): are we forgetting the pain

status: publishe

PENTA: development of a paediatric trauma registry in Flanders (Belgium)

status: publishe

Closing the gap: an audit of medical management in severe paediatric trauma

status: publishe

Assessing the level of consciousness in children: A plea for the Glasgow Coma Motor subscore

AIM: The Glasgow Coma Scale (GCS) is not always easy to score and its reliability has been questioned. In adults the GCS Motor score has proven a valuable alternative, as it is easier to assess yet shows similar predictive capacity for outcome. We wanted to test the non-inferiority of the Glasgow Coma Motor score GCS-M versus the Total score GCS-T for predicting outcome in children. MATERIALS AND METHODS: As part of the Flemish paediatric trauma registry (PENTA) we collected data on 96 consecutive children (0-18 years) with moderate to severe traumatic brain injury. Outcome was evaluated using a three level ordinal scale: [normal to mild disability, moderate to severe disability and death]. A number of proportional odds models were fitted for various choices of predictive variables (GCS-T, GCS-M, age, sex, and injury severity score ISS). For each model we calculated Somers'D(xy) rank correlation and NagelKerke's R(2)N index, both measures of the predictive performance of the model. RESULTS: All children had an injury to the brain that resulted in a hospital stay of more than 48h. Half of them had a "best" initial GCS of 15; 60%, a Motor score of 6. The median Injury Severity Score ISS was 16. Outcome was 'normal to mild' in 79 children, 'moderate to severe' in 7, and 'death' in 10. D(xy) values were 0.983 for the model with the Motor score and 0.972 for that with the total GCS, indicating excellent predictive performance for both. R(2)N indices were 0.862 and 0.813, respectively. Overall the difference between all models was small. CONCLUSION: The GCS Motor subscore was shown to have at least the same predictive ability for outcome as the total GCS. It is our opinion that the total GCS is unnecessarily complicated (especially in children). Using the Motor score alone will improve scoring compliance and statistical performance. We do not believe that the reduction in number of potential scores from 13 to 6 would decrease the descriptive capacity significantly, since clinical algorithms typically group values of the total GCS into five or fewer ranges.status: publishe