Aggregation of the neuroblastoma-associated mutant (S120G) of the human nucleoside diphosphate kinase-A/NM23-H1 into amyloid fibrils

Abstract

The human nucleoside diphosphate (NDP) kinase A, product of the NME1 gene also named NM23-H1, is known as a metastasis suppressor protein. A naturally occurring variant, S120G, identified in neuroblastomas, possesses native three-dimensional structure and enzymatic activity but displays reduced conformational stability and a folding defect with the accumulation of a "molten globule" folding intermediate during refolding in vitro. As such intermediate has been postulated to be involved in amyloid formation, NDP kinase A may serve as a model protein for studying the relationship between folding intermediates and amyloid fibrils. The NDP kinase A S120G was heated in phosphate buffer (pH 7.0). The protein precipitated as amyloid fibrils, as demonstrated by electron microscopy, Congo red, and thioflavin T binding and FTIR spectroscopy. The NDP kinase A S120G, at neutral pH and at moderate temperature experiences a transition towards amyloid fibrils. The aggregation process was faster if seeded by preformed fibrils. The fibrils presented a large proteinase K-resistant core not including residue Gly 120, as shown by mass spectrometry. This suggests that the aggregation process is triggered by the reduced stability of the S120G variant and not by a specific increase in the kinase domain intrinsic aggregation propensity at the place of mutation. This constitutes one of the few reports on a protein involved in cancer biology able to aggregate into amyloid structures under mild conditions.

Protein aggregation profile of the human kinome

“…Accordingly, we found that 85% of the human kinase domains include at least one amyloidogenic stretch and in many cases several of them. These data are in agreement with the observation that, in vitro , the population of partially unfolded states results in amyloid aggregation in different, unrelated, kinase domains (Damaschun et al, 2000; Agocs et al, 2010, 2012; Georgescauld et al, 2011). The mapping of these amyloidogenic regions on the three-dimensional structures of catalytic domains belonging to different human kinase groups indicates that, in most cases, they overlap with regular elements of secondary structure, providing support to the hypothesis that the molecular determinants responsible for amyloid formation coincide with those maintaining the native structure of proteins (Sabate et al, 2012).…”

“…Nevertheless, even those globular proteins selected to be highly soluble cannot avoid the presence of aggregation “sensitive” stretches in their sequences (Sabate et al, 2012). Accordingly, we have recently shown that a cancer associated point mutant of human nucleoside diphosphate kinase A displaying reduced conformational stability forms amyloid fibrils under close to physiological conditions (Georgescauld et al, 2011). Similarly, a partially folded intermediate of phosphoglycerate kinase has been shown to self-assemble into amyloid fibrils (Damaschun et al, 2000; Agocs et al, 2010).…”

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23‐H1 by the natural osmolyte trimethylamine‐N‐oxide

“…Similar stabilization of folding intermediates have previously been reported [22,23]. The tendency of the S120G-NDPK-HA to populate non-native folding intermediates is probably essential for its assembly in amyloid fibrils [24]. The human nucleoside diphosphate kinase A (product of the nm23-H1 gene) (NDPK-HA) (A) and S120G mutant of the human NDPK-HA (S120G-NDPK-HA) (B), native (full symbols) or unfolded in 9 M urea (empty symbols), were injected into a Superdex 75 column equilibrated with buffer A containing 0.2 M NaCl (circles) or buffer A containing 0.2 M NaCl plus 6 M urea and 2 M trimethylamine-N-oxide (TMAO) (squares).…”

“…The folding intermediates were often evoked in the recent literature as being essential during formation of amyloid fibrils [14,15]. We discovered that S120G mutant of the human NDPK-HA (S120G-NDPK-HA) assembles to amyloid fibrils by moderate heating [24]. Understanding the effect TMAO, on stability by MG is of great interest in this new and exciting perspective.…”

A critical evaluation of biochemical activities reported for the nucleoside diphosphate kinase/Nm23/Awd family proteins: opportunities and missteps in understanding their biological functions

“…NDPK-A/Nm23-H1 S120 is highly conserved throughout evolution and undergoes serine phosphorylation by casein kinase I (CKI), which is essential for the formation of the NDPK-A/Nm23-H1-Pn complex (Garzia et al 2008). As reported in this issue of Naunyn-Schmiedeberg's Archives of Pharmacology, a naturally occurring mutant of NDPK-A at S120, the S120G mutant, has the tendency to aggregate into amyloid structures (Georgescauld et al 2011). In addition to CKI, casein kinase II (CKII) might phosphorylate S120 in nm23-H1, too.…”