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PDB Reference: macrophage migration inhibitory factor, complex with phenethyl isothiocyanate, 4f2k.Macrophage migration inhibitory factor is irreversibly inhibited via covalent modification by phenethyl isothiocyanate, a naturally occurring compound with anti-inflammatory and anticancer properties. The structure of the modified protein obtained from X-ray diffraction data to 1.64 Å resolution is presented. The inhibitor sits within a deep hydrophobic pocket between subunits of the homotrimer and is highly ordered. The secondary structure of macrophage migratory inhibitory factor is unchanged by this modification, but there are significant rearrangements, including of the side-chain position of Tyr37 and the main chain of residues 31-34. These changes may explain the decreased binding of the modified protein to the receptor CD74. Together with the pocket, the areas of conformational change define specific targets for the design of more selective and potent inhibitors as potential therapeutics.

The regulation of Lp(a) clearance from circulation by cellular uptake remains enigmatic. While multiple receptors have been implicated in Lp(a) uptake, each receptor only partially accounts for this uptake, and no endocytic mechanism has yet been prescribed for Lp(a) internalisation into the cell. In this study, we define macropinocytosis as the endocytic pathway responsible for internalising Lp(a). In both liver and macrophage cells, Lp(a) uptake is dependent on extracellular calcium and is inhibited by amiloride or its derivative EIPA. The uptake of apo(a) alone is mediated by macropinocytosis, indicating the apo(a) protein component is a primary regulator of Lp(a) uptake. Importantly, we found that common antidepressants modulate Lp(a) macropinocytosis in cell-type dependent manners. In macrophages, the tricyclic antidepressant, imipramine and selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine and sertraline all inhibit Lp(a) uptake. In liver cells, imipramine and citalopram strongly stimulated Lp(a) uptake, while sertraline inhibited uptake. Imipramine and citalopram enhanced Lp(a) uptake by enhancing cell surface binding of Lp(a) rather than upregulating macropinocytosis per se, indicating that immobilisation of Lp(a) on the plasma membrane is an important regulatory step in Lp(a) macropinocytosis. As the liver is the major catabolic route for Lp(a), we dissected the functional consequences of imipramine and citalopram stimulated uptake in liver cells. Both drugs increased Lp(a) delivery to Rab11-positive recycling endosomes, but not late endosomes or lysosomes, resulting in increased apo(a) recycling into the cellular media. Given that free apo(a) is reported to undergo consistent proteolysis in the extracellular space or in circulation, these results support the potential for the already approved drugs, imipramine and citalopram, as promising therapeutics to lower Lp(a) levels. This approach displays special utility in the large group of people who suffer from depression and anxiety for whom these drugs are commonly prescribed.

A system based on cyanobacterial split inteins, SICLOPPs (Split Intein Circular Ligation of Proteins and Peptides), has been used to synthesise a small natively cyclic plant protein, kalata B1, and cyclised versions of the natively linear therapeutic peptides ziconotide and leconotide. The cyclic versions of these naturally linear peptides include linker sequences between their native termini to allow the correct tertiary structure to form. The native structure of each includes three disulphide bonds characteristic of knottins. Cyclic permutations of leconotide yielded different proportions of correctly spliced product, identifying an optimal splice site and revealing the influence of residues around the splice junction. The rate of splicing was manipulated to facilitate affinity purification prior to intein-mediated removal of the affinity tag.