scholarly journals A phosphorylation code regulates the multi-functional protein RETINOBLASTOMA-RELATED1 in Arabidopsis thaliana

2021 ◽  
Author(s):  
Jorge Zamora-Zaragoza ◽  
Katinka Klap ◽  
Jaheli Sánchez-Pérez ◽  
Jean-Philippe Vielle-Calzada ◽  
Ben Scheres
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Fate ◽  
Protein Interactions ◽  
Point Mutations ◽  
Protein Protein Interactions ◽  
Functional Protein ◽  
Developmental Context ◽  
Stem Cell Maintenance ◽  
Environmental Responses ◽  
Division Cell

The RETINOBLASTOMA-RELATED (RBR) proteins play a central role coordinating cell division, cell differentiation and cell survival within an environmental and developmental context. These roles reflect RBR ability to engage in multiple protein-protein interactions (PPIs), which are regulated by multi-site phosphorylation. However the functional outcomes of RBR phosphorylation in multicellular organisms remain largely unexplored. Here we test the hypothesis that phosphorylation allows diversification of RBR functions in multicellular context. Using a representative collection of transgenic loss- and gain of function point mutations in RBR phospho-sites, we analysed their complementation capacity in Arabidopsis thaliana root meristems. While the number of mutated residues often correlated to the phenotypic strength of RBR phospho-variants, phospho-sites contributed differentially to distinct phenotypes. For example, the pocket-domain has a greater influence on meristematic cell proliferation, whereas the C-terminal region associates to stem cell maintenance. We found combinatorial effects between the T406 phopspho-site with others in different protein domains. Moreover, a phospho-mimetic and a phospho-defective variant, both promoting cell death, indicate that RBR controls similar cell fate choices by distinct mechanisms. Thus, additivity and specificity of RBR phospho-sites fine tune RBR activity across its multiple roles. Interestingly, a mutation disrupting RBR interactions with the LXCXE motif suppresses dominant phospho-defective RBR phenotypes. By probing protein-protein interactions of RBR variants, we found that LXCXE-containing members of the DREAM complex constitute an important component of phosphorylation-regulated RBR function, but also that RBR participates in stress or environmental responses independently of its phosphorylation state. We conclude that developmental-related, but not stress- or environmental-related functions of RBR are defined and separable by a combinatorial phosphorylation code.

scholarly journals Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

2017 ◽  
Vol 114 (11) ◽  
pp. E2146-E2155 ◽  
Author(s):  
Chi-Yun Lin ◽  
Johan Both ◽  
Keunbong Do ◽  
Steven G. Boxer
Keyword(s):  
Quantum Yield ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Photoactive Yellow Protein ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Low Efficiency ◽  
Green Fluorescent ◽  
Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

scholarly journals Network diffusion on multiple-layers: current approaches and integrative analysis of Rheumatoid Arthritis data.

2017 ◽  
Author(s):  
Noemi Di Nanni ◽  
Matteo Gnocchi ◽  
Marco Moscatelli ◽  
Luciano Milanesi ◽  
Ettore Mosca
Keyword(s):  
Rheumatoid Arthritis ◽  
Protein Interactions ◽  
Single Layer ◽  
Joint Analysis ◽  
Protein Protein Interactions ◽  
Functional Protein ◽  
Detection Function ◽  
Module Detection ◽  
Rheumatoid Arthritis Data ◽  
Network Diffusion

Network Diffusion has been proposed in several applications, thanks to its ability of amplifying biological signals and prioritizing genes that may be associated with a disease. Not surprising, the success of Network Diffusion on a “single layer” led to the first approaches for the joint analysis of multi-omics data. Here, we review integrative methods based on Network Diffusion that have been proposed with several aims (e.g. patient stratification, module detection, function prediction). We used Network Diffusion to analyse, in the context of physical and functional protein-protein interactions, genetic variation, DNA methylation and gene expression data from a study on Rheumatoid Arthritis. We identified functionally related genes with multiple alterations.

scholarly journals Competition NMR for Detection of Hit/Lead Inhibitors of Protein–Protein Interactions

Molecules ◽  
2020 ◽  
Vol 25 (13) ◽  
pp. 3017 ◽  
Author(s):  
Bogdan Musielak ◽  
Weronika Janczyk ◽  
Ismael Rodriguez ◽  
Jacek Plewka ◽  
Dominik Sala ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Point Mutations ◽  
Protein Protein Interactions ◽  
Competition Assay ◽  
Fragment Screening ◽  
Different Types ◽  
Lead Inhibitors

Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

scholarly journals Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

2006 ◽  
Vol 173 (4) ◽  
pp. 533-544 ◽  
Author(s):  
Chad D. Knights ◽  
Jason Catania ◽  
Simone Di Giovanni ◽  
Selen Muratoglu ◽  
Ricardo Perez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Biological Activities ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
P53 Gene ◽  
Protein Protein Interactions

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.

scholarly journals Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression

BMC Genomics ◽  
2009 ◽  
Vol 10 (1) ◽  
pp. 288 ◽  
Author(s):  
Stefanie De Bodt ◽  
Sebastian Proost ◽  
Klaas Vandepoele ◽  
Pierre Rouzé ◽  
Yves Van de Peer
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Ontology ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals BBX32 Interacts with AGL24 Involved in Flowering Time Control in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

2018 ◽  
Vol 47 (1) ◽  
pp. 34-45
Author(s):  
Guan-Peng MA ◽  
Da-Qin ZHAO ◽  
Tian-Wen WANG ◽  
Lin-Bi ZHOU ◽  
Gui-Lian LI
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Clock ◽  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Time Control ◽  
Flowering Time Control ◽  
Molecular Regulatory Mechanisms

B-box (BBX) zinc finger proteins play critical roles in both vegetative and reproductive development in plants. Many BBX proteins have been identified in Arabidopsis thaliana as floral transition regulatory factors, such as CO, BBX7 (COL9), BBX19, and BBX32. BBX32 is involved in flowering time control through repression of COL3 in Arabidopsis thaliana, but it is still elusive that whether and how BBX32 directly interacts with flowering signal integrators of AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Chinese cabbage (Brassica rapa L. ssp. pekinensis) or other plants. In this study, B-box-32(BBX32), a transcription factor in this family with one B-box motif was cloned from B. rapa, acted as a circadian clock protein, showing expression changes during the circadian period. Additional experiments using GST pull-down and yeast two-hybrid assays indicated that BrBBX32 interacts with BrAGL24 and does not interact with BrSOC1, while BrAGL24 does interact with BrSOC1. To investigate the domains involved in these protein-protein interactions, we tested three regions of BrBBX32. Only the N-terminus interacted with BrAGL24, indicating that the B-box domain may be the key region for protein interaction. Based on these data, we propose that BrBBX32 may act in the circadian clock pathway and relate to the mechanism of flowering time regulation by binding to BrAGL24 through the B-box domain. This study will provide valuable information for unraveling the molecular regulatory mechanisms of BrBBX32 in flowering time of B. rapa.

scholarly journals Crystal Structure of SEDL and Its Implications for a Genetic Disease Spondyloepiphyseal Dysplasia Tarda

2002 ◽  
Vol 277 (51) ◽  
pp. 49863-49869 ◽  
Author(s):  
Se Bok Jang ◽  
Yeon-Gil Kim ◽  
Yong-Soon Cho ◽  
Pann-Ghill Suh ◽  
Kyung-Hwa Kim ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Genetic Disease ◽  
Point Mutations ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Spondyloepiphyseal Dysplasia ◽  
Multiple Protein ◽  
Protein Particle ◽  
Eukaryotic Organisms ◽  
Spondyloepiphyseal Dysplasia Tarda

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in theSEDLgene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 Å resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

scholarly journals Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4381-4390 ◽  
Author(s):  
Wendy R. Gordon ◽  
Monideepa Roy ◽  
Didem Vardar-Ulu ◽  
Megan Garfinkel ◽  
Marc R. Mansour ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Integrity ◽  
Crystal Packing ◽  
Novel Mutation ◽  
Lymphoblastic Leukemia ◽  
Regulatory Region ◽  
Point Mutations ◽  
Protein Protein Interactions ◽  
Proteolytic Resistance ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the 3 Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T-cell acute lymphoblastic leukemia lymphoma (T-ALL). Here, we report the x-ray structure of the Notch1 NRR in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in intermolecular or intramolecular protein-protein interactions. The majority of known T-ALL–associated point mutations map to residues in the hydrophobic interior of the Notch1 NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.

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