scholarly journals Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 593
Author(s):  
Seav-Ly Tran ◽  
Delphine Cormontagne ◽  
Jasmina Vidic ◽  
Gwenaëlle André-Leroux ◽  
Nalini Ramarao
Keyword(s):  
Cell Wall ◽  
Protein Interactions ◽  
Health Concern ◽  
Protein Protein Interactions ◽  
Src Homology 3 ◽  
Intrinsically Disordered ◽  
Food Borne ◽  
A Cell ◽  
Src Homology

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b1–3) likely to control protein–protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b3 and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

scholarly journals A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2

2021 ◽  
Vol 7 (15) ◽  
pp. eabg1950
Author(s):  
Nicolas Bery ◽  
Carole J.R. Bataille ◽  
Angela Russell ◽  
Angela Hayes ◽  
Florence Raynaud ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Binding Sites ◽  
Screening Method ◽  
Chromosomal Translocation ◽  
Resonance Method ◽  
Protein Protein Interactions ◽  
Intracellular Antibody ◽  
A Cell

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody–guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

scholarly journals Coordination of RNA and protein condensation by the P granule protein MEG-3

2020 ◽  
Author(s):  
Helen Schmidt ◽  
Andrea Putnam ◽  
Dominique Rasoloson ◽  
Geraldine Seydoux
Keyword(s):  
Protein Interactions ◽  
Intrinsically Disordered Protein ◽  
Protein Protein Interactions ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
C Terminus ◽  
Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

scholarly journals The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel-Lindau tumor suppressor

2020 ◽  
Author(s):  
Antonella Falconieri ◽  
Giovanni Minervini ◽  
Raissa Bortolotto ◽  
Damiano Piovesan ◽  
Raffaele Lopreiato ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Protein Interactions ◽  
Hypoxia Inducible Factor ◽  
Protein Protein Interactions ◽  
Von Hippel Lindau ◽  
Intrinsically Disordered ◽  
Tetramerization Domain ◽  
Specific Association ◽  
Cycle Regulation

AbstractMutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1-α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). Importantly, we found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already proposed for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell proliferation, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data support the idea that pVHL30 plays a role in MDM2 regulation, suggesting a wider interplay among hypoxia sensing and cell cycle regulation.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

Insights into the structure and protein–protein interactions of the pro-apoptotic protein ASPP2

2007 ◽  
Vol 35 (5) ◽  
pp. 966-969 ◽  
Author(s):  
S. Rotem ◽  
C. Katz ◽  
A. Friedler
Keyword(s):  
Intermolecular Interactions ◽  
Protein Interactions ◽  
Intramolecular Interaction ◽  
Protein Protein Interactions ◽  
Sh3 Domains ◽  
Src Homology 3 ◽  
Rich Domain ◽  
C Terminus ◽  
Apoptotic Protein ◽  
Src Homology

ASPP (apoptosis-stimulating protein of p53) 2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. Here, we provide an overview of the structure and protein–protein interactions of ASPP2. The C-terminus of ASPP2 contains Ank (ankyrin) repeats and an SH3 domain (Src homology 3 domain). The Ank–SH3 domains mediate interactions between ASPP2 and numerous proteins involved in apoptosis such as p53 and Bcl-2. The proline-rich domain of ASPP2 is unfolded in its native state, but was not shown to mediate intermolecular interactions. Instead, it makes an intramolecular domain–domain interaction with the Ank–SH3 C-terminal domains of ASPP2. This intramolecular interaction between the unstructured proline-rich domain and the structured Ank–SH3 domains in ASPP2, which is possible due to the unfolded nature of the proline-rich domain, is proposed to have an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins.

scholarly journals Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

2018 ◽  
Author(s):  
Ryo Hayama ◽  
Mirco Sorci ◽  
John J. Keating ◽  
Lee M. Hecht ◽  
Joel L. Plawsky ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Binding Mode ◽  
Nucleocytoplasmic Transport ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Mutant Variant

ABSTRACTProtein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

scholarly journals Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2

2019 ◽  
Vol 5 (11) ◽  
pp. eaav9810 ◽  
Author(s):  
Min Young Kim ◽  
Insung Na ◽  
Ji Sook Kim ◽  
Seung Han Son ◽  
Sungwoo Choi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Cancer Metastasis ◽  
Epithelial Mesenchymal Transition ◽  
Tumor Model ◽  
Intrinsically Disordered Protein ◽  
Protein Protein Interactions ◽  
Mesenchymal Transition ◽  
Intrinsically Disordered

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

scholarly journals AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kohki Kido ◽  
Satoshi Yamanaka ◽  
Shogo Nakano ◽  
Kou Motani ◽  
Souta Shinohara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
De Novo ◽  
Reconstruction Algorithm ◽  
Target Protein ◽  
Protein Protein Interactions ◽  
Key Technology ◽  
A Cell

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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