Interleukin 32 (IL-32) contains a typical α-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1.
IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and α-helixes, but no β sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32β- or IL-32γ-induced cytotoxicity mediated through caspase-3. Although IL-32α interacted with the extracellular part of αVβ3 and αVβ6 integrins, only the αVβ3 binding was inhibited by small RGD peptides. Additionally, IL-32β was able to bind to αVβ3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct α-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32β- or IL-32γ-induced cytotoxicity. Interestingly, IL-32α binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32α/paxillin binding, whereas FAK also could interact with IL-32α, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling.
Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, 6500 HB, Nijmegen, The Netherlands.
Bas
B
Heinhuis
Marije I
MI
Koenders
Wim B
WB
van den Berg
Mihai G
MG
Netea
Charles A
CA
Dinarello
Leo A B
LA
Joosten
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D019295:Computational Biology; D004789:Enzyme Activation / Q000187:drug effects; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D007378:Interleukins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D051419:Paxillin / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects; D012984:Software
D000951:Antigens, Neoplasm; C496971:IL32 protein, human; D039302:Integrin alphaVbeta3; D016023:Integrins; D007378:Interleukins; D009842:Oligopeptides; D051419:Paxillin; D020033:Protein Isoforms; C073878:integrin alphavbeta6; C047981:arginyl-glycyl-aspartic acid; D051417:Focal Adhesion Kinase 1; D053148:Caspase 3