FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

Chakraborty, Sandeep; Núñez, David; Hu, Shih-Yang; Domingo, María Pilar; Pardo, Julián; Karmenyan, Artashes; Gálvez, Eva M.; Chiou, Arthur

doi:10.1371/journal.pone.0102572

Cited by 16 publications

(12 citation statements)

References 63 publications

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“…We conducted FRET measurements following the experimental procedure described by Chakraborty et al [ 20 ] based on the theoretical and experimental procedure developed by Song et al [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

et al. 2015

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The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG). For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.

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Section: Methodsmentioning

confidence: 99%

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

et al. 2015

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“…Given this fact, the monitored energy transfer is an average dominated by the closest tags. We chose to use Alexa Fluor 488 as FRET donor and Alexa Fluor 555 as FRET acceptor, a conjugate couple largely used (29,30,31). FRET between this fluorophore pair is efficient in this context since the distance for 50% FRET (Förster distance, R o ) is 7.0 nm, compatible with the range 2.7 to 7.0 nm indicated above.…”

Section: Labeling Of H and L Ferritin Homopolymers For Fret Analysismentioning

confidence: 99%

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Carmona

Poli

Bertuzzi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background: The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H-and Ltype that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. Methods: We used the Fluorescence Resonance Energy Transfer (FRET) by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. Results: The FRET-efficiency increase from <0.3 of the disassembled to >0.7 in the reassembled allowed to study the self-assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. Conclusion: Heterodimeric (H/L) subunit association is preferred during H/L heteropolymers formation. The H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize and the ferroxidase activity of the heteropolymer reaches a plateau. General significance: This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Keywords

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“…A metal ion-dependent adhesion site (MIDAS) is located in the upper face of the I-domain ( 18 ). ICAM-1 interacts with LFA-1 through the binding of its first Ig-domain (D 1 ) with the MIDAS within the I-(inserted) domain at the top of the N-terminus of αL subunit of LFA-1 ( 19 ). The ICAM-1/LFA-1 interaction is facilitated by magnesium and manganese divalent cations, assorted with five amino acids of MIDAS in LFA-1 and glutamate in domain 1 of ICAM-1 ( 20 ).…”

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confidence: 99%

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

et al. 2017

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The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

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FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

Cited by 16 publications

References 63 publications

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

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