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  • br Material and methods br Results

    2022-08-03


    Material and methods
    Results
    Discussion Functional characterization of naturally occurring GCK mutations in patients with impaired glucose homeostasis has widened our knowledge of the catalytic and regulatory mechanisms of this enzyme [3]. In this work, we have biochemically characterized a group of GCK-MODY mutations within the α8-helix containing a nuclear export signal. α8-helix is found on the surface of the large domain of glucokinase, distant from the deep cleft that hosts the active site [6] (Fig. 8). We would predict that mutations V302E, L304P, L306R and L309H cause structural changes in the α8-helix by introducing polar residues in a hydrophobic environment and because of steric conflicts with neighbouring residues (Fig. 8). Indeed, we found that mutations V302E, L304P, L306R and L309H inactivate GST-GCK enzymatic activity by reducing strongly Kcat values and slightly glucose affinity, and likely decreasing protein stability, as suggested by the thermal inactivation and reduced purification yield of most of the recombinant mutant proteins in vitro. These findings are consistent with previous results showing that mutation L304P induced faster GCK-protein degradation in MIN6 Minocycline HCl [32]. Moreover, mutations E300Q, E300K, R303W, R308W and L309P have also been shown to decrease protein stability [25,[33], [34], [35], [36], [37]]. Taken together, these data indicate that α8-helix is important for glucokinase stability and kinetics. Transport of proteins larger than the exclusion limit size (≈40 kDa) through the nuclear pore is mediated by importins and exportins, which recognize nuclear localization signals (NLS) and NES, respectively, in the cargo protein [38]. Although a GCK NLS has been recently reported to be functional in pancreatic beta-cells, GCK nuclear import in hepatocytes depends on its interaction with GKRP [17,39]. Previous analysis of the crystal structure of the GCK-GKRP complex indicates that GCK α8-helix is not directly involved in the interaction with GKRP [15,11] (Fig. 8c). Accordingly, we found that most of the GCK mutants analysed here are inhibited in vitro by recombinant human GKRP, do interact with the regulatory protein in the yeast two–hybrid system and are translocated to the nucleus when co-expressed with GKRP in cultured cells. Exceptions are GCK mutants L306R and L309P, which are not detected in the cell nucleus even when GKRP was overexpressed. Consistently, mutation L306R prevents GCK binding to GKRP in yeast two-hybrid. Similar results have been reported for GCK mutant L309R [40]. Substitutions of hydrophobic leucine residues by basic arginine in the α8-helix may induce GCK folding defects, thus preventing recognition by GKRP. Surprisingly, mutant L309P still interacts with the regulatory protein in yeast two-hybrid. We found that leptomycin B does not induce the nuclear accumulation of GFP-GCK(L309P) (results not shown) and that mutation L309P does not activate the GCK-NES, ruling out the possibility of a more active nuclear export resulting in the cytoplasmic accumulation of this mutant. We found that some HepG2 cells expressing GFP-GCK(L309P) retain GKRP-mCherry in the cytoplasm, thus supporting the idea that these two proteins interact but cannot be imported to the nucleus. One possible explanation is that mutation L309P impairs the conformational adjustment of the GCK-GKRP dimer [41] or that the mutant GCK-GKRP complex is not recognized by the import machinery. Glucokinase contains a functional NES (300ELVRLVLLKLV310) that fits the NES consensus class 1b for the major export receptor exportin1/CRM1 (ϕ1-X2-ϕ2-X2-ϕ3-X-ϕ4, where ϕ is a hydrophobic residue, mainly Leu, but also Ile, Val, Phe or Met, and X might be any aminoacid) [17,42,43]. GKRP is required for GCK nuclear import but not for its nuclear export [17,18]. Since some of the mutations in GCK α8-helix impair its nuclear import, we used a previously developed method [17] to analyse the specific effect of these mutations on GCK-NES activity. This approach uses a small GFP-fusion protein (34 kDa) containing residues 299–359 of rat glucokinase, which can diffuse freely both into and out of the nucleus and thus does not require GKRP-dependent import. In agreement with previous work [17], we found that GCK-NES (residues 300 to 310) mediates nuclear export in our cultured cells and that NES activity is impaired in the mt3 control mutant. We further demonstrated that GCK-NES function requires exportin1/CRM1 by showing that nuclear export of the GFP-fusion protein is inhibited by leptomycin B and enhanced upon CRM1 overexpression. CRM1-interacting NES are diverse but share common characteristics such as a low affinity for CRM1 and hydrophobic residues that bind a hydrophobic groove in CRM1 [42,44,45]. Our mutational analysis has uncovered some functional and structural features of the GCK-NES. The MODY2 mutations L304P, L309H and L309P affect hydrophobic residues ϕ2 and ϕ4, while mutations E300K, E300Q, V302E, R303W and K308W (rat K308W corresponds to human R308W mutation) affect spacer positions. Once bound to CRM1, many NESs adopt an amphipathic α-helix conformation at the N-terminus (ϕ1–ϕ3), and have a more relaxed structure at the C-terminus (ϕ3–ϕ4) [44,45]. This may explain why proline substitution at ϕ2 position has a stronger inhibitory effect than at ϕ4. Indeed, mutations L309P produces just a slight decrease in export activity, whereas mutation L304P strongly inactivates the NES. Spacer residues are also important for NES activity [44,46]. For instance, bulky tryptophan residues are rarely found at the C-termini of NES [46]. The substitutions of basic residues to tryptophan in R303W and K308W mutants produce different effects. The R303W substitution between ϕ1 and ϕ2 disturbs the N-terminal amphipathic helix and, as expected, results in a weaker NES. In contrast, the K308W substitution between ϕ3 and ϕ4 induces a perinuclear distribution of the GFP fusion protein. Interestingly, substitution at the same position in a synthetic NES has been shown to produce the same localization pattern [47]. Such NES, referred as supraphysiological, strongly interacts with CRM1 and impair nuclear export by blocking export complexes at nuclear pores. Previous work reported that acidic residues are found at relative high frequency at spacer positions and that substitution of neutral residues flanking ϕ1 to glutamate enhance CRM1 binding [44,46,48]. However, the negative effect of mutation V302E on export activity and the positive effect of mutations E300K and E300Q rather suggests that the presence of Glu next to ϕ1 weakens GCK-NES activity. We hypothesize that Glu300 contributes to the low affinity of the NES for CRM1, which is required for its proper functioning. In summary, our results support the idea that spacer residues of the GCK-NES modulate its activity either negatively or positively, thereby contributing to the fine-tuning of its function.