A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants

Abstract

Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation

“…The overall functionality of CCS1 is significantly more complex than that of a standard metallo-chaperone. Related work on the human form of CCS1 has shown an ability to stabilize select disease-causing SOD1 mutants, although any role in SOD1-dependent ALS is unclear (18). Our results support a model where initial binding of immature SOD1 by CCS1 facilitates site-appropriate Zn binding by stabilizing liganding residues present in the disulfide loop of SOD1 (Fig.…”

“…Work by our group and others supports a model where CCS1 possesses both Cu and molecular chaperoning roles that are impeded at specific points in the maturation process by pathogenic ALS mutants (17,19,25,39). However, related studies have purported that molecular chaperone-like functions of CCS1 actually work to target and stabilize disease-causing forms of SOD1 (18,40). Nevertheless, a detailed biochemical evaluation of these newly discovered molecular chaperoning functions of CCS1 is severely lacking.…”

“…The Cu chaperone for SOD1 (CCS1) mediates at least two of these modifications (14) by engaging nascent SOD1, inserting a Cu ion, and directing disulfide bond formation (15,16). However, increasing evidence supports additional roles for CCS1 during the SOD1 activation process (17)(18)(19). Metallo-chaperones are target-specific, as CCS1 does not recognize other metallo-proteins (reviewed in Ref.…”

“…Here, CCS1 D3 simply functions as a disulfide isomerase responsible for the oxidation of the SOD1 disulfide bond separately from Cu delivery. Related work from this group points to molecular chaperone-like role(s) for CCS1 upon binding to variant forms of SOD1 (18). The mechanism of SOD1 maturation by CCS1 may not be universal, as further evidenced by the conditional requirement for D1 in yeast (20), but not humans (32), whereas other organisms have CCS1 molecules that lack the MXCXXC motif in D1 (33) or even CCS1 at all (34).…”

iNEXT: a European facility network to stimulate translational structural biology

“…This approach leverages existing mammalian protein expression technologies to allow the proteins of interest to be directly expressed and isotope‐labelled within the cells (Fig. ), and is especially useful to characterize protein folding and maturation , cofactor binding, redox state changes and other physiological processes occurring after protein synthesis. An in‐organello NMR approach was also developed, in which protein expression was targeted to mitochondria via a specific targeting sequence .…”