Direct Modification of the Proinflammatory Cytokine Macrophage Migration Inhibitory Factor by Dietary Isothiocyanates

Abstract

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.Isothiocyanates are a class of phytochemicals with recognized anti-cancer activity. They can act in a chemopreventive capacity via inhibition of carcinogen-activating phase I enzymes (1) and induction of phase II detoxification enzymes (2). Isothiocyanates are also active in the post-initiation phase of tumorigenesis and are, therefore, proposed to have chemotherapeutic potential (3, 4). Isothiocyanate-mediated disruption of cancer progression is achieved by a variety of mechanisms including modulation of cell growth (5), inhibition of angiogenesis (6), suppression of metastasis (7), and induction of apoptosis (8, 9). Isothiocyanates can also modulate inflammatory pathways via inhibition of the transcription factor nuclear factor B (10).The electrophilic carbon residue in the isothiocyanate moiety (-NACAS) is capable of reacting with biological nucleophiles such as cysteine in proteins and the tripeptide glutathione (11,12). Binding of isothiocyanates to Kelch-like ECH-associated protein 1 (Keap1) (13), transient receptor potential channels (14), MEKK1 protein kinase (15), and tubulin (16) has been demonstrated to occur via covalent modification of cysteine. Reaction with amines to form stable thiourea derivatives can also occur. However, this is generally considered to be a less favorable reaction at physiological pH (11).To elucidate the major cellular targets of biologically active isothiocyanates, we have utilized an affinity-based target identification approach. An amine linker was added to phenethyl isothiocyanate (PEITC) 3 without compromising cytotoxicity, and the molecule was immobilized to a solid phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was identified as a major biological target of PEITC. Using mass spectrometry and site-directed mutagene...

Electrophilic tuning of the chemoprotective natural product sulforaphane

“…Using a proteomics approach, we have discovered at least six protein targets of 8f other than Keap1, MIF, AKAP149, Prx3, Trx, HAT1, and KSRP. Inhibition of MIF, a well-established chemokine (46), has previously been associated with a derivative of sulforaphane, phenethyl isothiocyanate (47), and may be important in blocking inflammation. Modification of AKAP149 by 8f could influence protein kinase cell signaling, whereas modification of Prx3 and Trx could affect the antioxidant response.…”

Neuroprotective Effects of Sulforaphane after Contusive Spinal Cord Injury

“…[20][21][22][23] Sulforaphane and other ITCs inactivate the tautomerase activity of MIF by reacting with a highly nucleophilic N-terminal proline residue in the active site of the enzyme. 41 Recent studies have demonstrated MIF tautomerase activity in human urine, and that oral administration of SF-rich broccoli sprout extracts to volunteers almost completely abolished this activity. 19 To our knowledge, urinary MIF tautomerase activity and the effects of SCI on urinary MIF activity have not previously been examined in rodents.…”

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

“…52 Interestingly, studies from our group have shown that only catalytically active trimers undergo modification by these compounds at Pro1 in vitro and in the cell. 52,58 These studies highlight the reactivity of the N-terminal proline, Pro1, and provide a basis of novel strategies for specific detection and quantification of catalytically active MIF and targeting of its catalytic activities. The enrichment factor for the in silico protocol combining three parallel strategies was increased by a factor of 11.43 compared to the Maybridge experimental screening.…”