Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human <scp>phosphomannomutase 2</scp> structures

Briso-Montiano, Álvaro; Caño-Ochoa, Francisco Del; Vilas, Alicia; Velázquez‐Campoy, Adrián; Rubio, Vicente; Pérez, Belén; Ramón‐Maiques, Santiago

doi:10.1002/jimd.12461

Cited by 8 publications

(22 citation statements)

References 57 publications

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“…We chose to focus on sec53 -V238M and sec53 -F126L because they are two of the most common disease alleles in humans and because the mutations have distinct and well-characterized effects on protein structure and function (Figure 1A) (Andreotti et al, 2015; Briso-Montiano et al, 2022; Citro et al, 2018; Kjaergaard et al, 1999; Pirard et al, 1999; Silvaggi et al, 2006). We evolved 96 haploid populations of each mutant sec53 genotype (p SEC53-sec53-V238M , pACT1-sec53-V238M, pACT1-sec53-F126L ) and 48 haploid populations of each wild-type SEC53 genotype (p SEC53 - SEC53 -WT, pACT1-SEC53-WT ) for 1,000 generations in rich glucose medium (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

Experimental evolution of phosphomannomutase-deficient yeast reveals compensatory mutations in a phosphoglucomutase

Vignogna

Allocca

Monticelli

et al. 2022

Preprint

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The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N-linked glycosylation. The yeast gene SEC53 encodes a nearly-identical homolog of human PMM2. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53-V238M and sec53-F126L, or wild-type SEC53. We find that after 1,000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease associated alleles.

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Section: Resultsmentioning

confidence: 99%

Experimental evolution of phosphomannomutase-deficient yeast reveals compensatory mutations in a phosphoglucomutase

Vignogna

Allocca

Monticelli

et al. 2022

Preprint

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“…The expression plasmid pDEST17-D18 (Source BioScience) encoding human PMM2 cDNA (NM_000303.2), the pReceiver-B01 plasmid encoding mouse Pmm2 cDNA (NM_016881.3), and the pOPINB expression vector (Briso-Montiano et al, 2021), all with an N-terminal His 6 -tag, were used to transform Escherichia coli BL21Star TM DE3 One-Shot Cells (Invitrogen). PMM2 pathogenic variants were introduced by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara) and specially designed primers.…”

Section: Wild-type (Wt) Pmm2 and Pathogenic Variant Gene Expressionmentioning

confidence: 99%

“…Cells were harvested by centrifugation. Protein purification and concentration were performed as previously described (Briso-Montiano et al, 2021).…”

Section: Wild-type (Wt) Pmm2 and Pathogenic Variant Gene Expressionmentioning

confidence: 99%

“…The selected variants were present in 27 different genotypes. Loss-of-function variants (i.e., premature stop codons, splicing variants) or severe missense variants, such as p.Arg141His or p.Arg123Gln (Briso-Montiano et al, 2021;Yuste-Checa et al, 2015), were not included (Supporting Information: Table S1). Two variants, p.Phe119Leu and p.Ile120Thr, located in the PMM2 dimerization region, and one variant, p.Asn216Asp, identified in an Australian screening program (neither of which have been detected in the Spanish population) were also included.…”

Section: Selection Of Variantsmentioning

confidence: 99%

“…(Briso- Montiano et al, 2021). Whereas p.Ile120Thr could not be expressed, both p.Glu93Ala or p.Phe119Leu hampered protein dimerization (Figure2a,c), and the mutated proteins in solution were in an equilibrium between monomeric and dimeric forms irresolvable by SEC.…”

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confidence: 99%

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A functional platform for the selection of pathogenic variants of PMM2 amenable to rescue via the use of pharmacological chaperones

et al. 2022

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Different strategies are being investigated for treating PMM2-CDG, the most common congenital disorder of glycosylation. The use of pharmacochaperones (PCs) is one of the most promising. The present work characterizes the expression, stability, and enzymatic properties of 15 previously described clinical variants of the PMM2 protein, four novel variants, the Pmm2 mouse variant p.Phe115Leu, and its p.Phe119Leu human counterpart, with the aim of extending the potential use of pharmacochaperoning treatment. PMM2 variants were purified as stable homodimers, except for p.Asp65Gly, p.Ile120Thr, and p.Thr237Lys (no expression detected), p.Thr226Ser and p.Val231Met (aggregates), and p.Glu93Ala, p.Phe119Leu, and p.Phe115Leu (partial dissociated).Enzyme activity analyses identified severe variants and milder ones. Pure dimeric mutant proteins showed a reduction in thermal stability except for p.Asn216Asp. The thermal stability of all the unstable mutants was recovered in the presence of the PC compound VIII. This study adds to the list of destabilizing human variants amenable to rescue by small chemical compounds that increase the stability/activity of PMM2. The proposed platform can be reliably used for assessing the disease-causing effects of PMM2 missense variants, for assessing the correlation between genotype and phenotype, for confirming new clinical defects, and for identifying destabilizing mutations amenable to rescue by PCs.

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Cysteine mutations impair the structural stability of phosphomannomutase 2 (PMM2) in glycosylation-associated metabolic disorders

Pan

Lin

et al. 2023

Genes & Diseases

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Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures

Cited by 8 publications

References 57 publications

Experimental evolution of phosphomannomutase-deficient yeast reveals compensatory mutations in a phosphoglucomutase

Experimental evolution of phosphomannomutase-deficient yeast reveals compensatory mutations in a phosphoglucomutase

A functional platform for the selection of pathogenic variants of PMM2 amenable to rescue via the use of pharmacological chaperones

Cysteine mutations impair the structural stability of phosphomannomutase 2 (PMM2) in glycosylation-associated metabolic disorders

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