Functional analysis of tumour necrosis factor-α-related apoptosis-inducing ligand (TRAIL): cysteine-230 plays a critical role in the homotrimerization and biological activity of this novel tumoricidal cytokine

Abstract

We have determined that the mutation of the cysteine-230 residue to either glycine or serine in TRAIL (tumour necrosis factor-alpha-related apoptosis-inducing ligand) results in the formation of a structurally incompetent dimer and a consequent loss of apoptotic activity. Similarly, chemical modification of the thiol residues present in both reduced and Zn(2+)-depleted trimer converts TRAIL into an inactive dimer. We postulate that cysteine-230 plays a critical role in homotrimerization of this tumoricidal cytokine.

Inducing rigid local structure around the zinc-binding region by hydrophobic interactions enhances the homotrimerization and apoptotic activity of zinc-free TRAIL

“…TRAIL C230A exhibited only 4.4-fold lower affinity with TR2 than wild-type TRAIL, but it did not form a stable trimeric complex and exhibited no apoptotic activity, consistent with previous observations [6,[9][10][11]. Alternative substitutions of Cys230 with Ser, Val, Ile, Met, Leu, Thr, or His gave the same results (data not shown).…”

“…2 and Table 1). Even though the exact homotrimerization constant is not available for wild-type TRAIL, previous SEC and crystallographic studies have shown that wild-type TRAIL exists as a stable trimer in solution, down to at least micromolar concentrations [5,6,11,12]. As expected, wild-type TRAIL was eluted as a trimer with the expected MW of 60 kDa ( 20 kDa · 3) (Fig.…”

“…2B). Changes in elution buffers with different pH and ionic strength or SEC columns with different resins gave the same result (data not shown) [3,11]. A previous report showed that TRAIL mutant with the replacement of Cys230 with Ser, was barely detected as the monomeric form by SEC [9].…”

“…The buried Cys230 residues coordinate a zinc ion (Zn 2+ ) in conjunction with a chloride ion, resulting in a unique tetrahedral zinc-binding site in TRAIL trimer [6]. Numerous studies of TRAIL with substitutions of Cys230 with other residues of Ala, Ser or Gly resulted in significant loss of the quaternary structure and the bioactivity of inducing apoptotic cell death [6,[9][10][11]. Further, zinc-depleted TRAIL has lower thermodynamic stability and solubility to be easily converted into biologically inactive dimeric and/or monomeric forms and aggregated species [5][6][7]9,10].…”

Cancer gene therapy using a novel secretable trimeric TRAIL

“…Oxidation between cysteine residues forms a homodimeric TRAIL. 24 As we and others demonstrated recently, 24,25 the dimeric TRAIL has much lower apoptotic activity than trimeric TRAIL. These data indicate that trimerization is critical for proper TRAIL function.…”