Cooperative energetic effects elicited by the yeast Shwachman-Diamond syndrome protein (Sdo1) and guanine nucleotides modulate the complex conformational landscape of the elongation factor-like 1 (Efl1) GTPase

Luviano, Axel; Cruz-Castañeda, Roberto; Sánchez-Puig, Nuria; García‐Hernández, Enrique

doi:10.1016/j.bpc.2019.02.003

Cited by 8 publications

(20 citation statements)

References 68 publications

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“…The association of the large 60S ribosomal subunit with the small 40S subunit is sterically blocked by eIF6. The release of eIF6 allows the joining of the 60S and 40S subunits and the formation of the translationally active 80S ribosome [4,20]. In addition, SBDS plays a role in the conformational maturation of the ribosomal P-site [19].…”

Section: Introductionmentioning

confidence: 99%

Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman–Diamond Syndrome Subjects

Frattini

Bolamperti

Valli

et al. 2021

IJMS

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Shwachman–Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.

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Section: Introductionmentioning

confidence: 99%

Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman–Diamond Syndrome Subjects

Frattini

Bolamperti

Valli

et al. 2021

IJMS

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“…CD spectra of purified rHuGM-CSF in water were recorded at 37 °C in the far-UV region with a JASCO J-720 spectropolarimeter (Jasco Inc., Easton, MD) described elsewhere (Luviano et al 2019 ). Protein solutions of ~ 0.05 mg/ml were loaded into a quartz cell of 0.1-cm length path.…”

Section: Methodsmentioning

confidence: 99%

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli

Restrepo-Pineda

Sánchez-Puig

Pérez³

et al. 2022

Appl Microbiol Biotechnol

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The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte–macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR -cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs. Key Points • Pre-induction temperature determines inclusion bodies architecture • In pre-induction (above 34 °C), the heat shock response increases recombinant protein production • Inclusion bodies at higher pre-induction temperature show a lower amyloid content Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11908-z.

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“…S. cerevisiae Efl1 wild type and mutants R1086Q, L910K, T33A, and R1086Q P151L were recombinantly expressed in S. cerevisiae and purified as described elsewhere [ 6 , 17 , 26 ]. Protein purity was assessed with SDS-PAGE, and the samples were stored in 50 mM HEPES-KOH pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , and 10% glycerol at −80 °C until further use.…”

Section: Methodsmentioning

confidence: 99%

“…EFL1 is a molecular motor whose conformational plasticity is central for the release of eIF6 and is finely regulated by interactions with SBDS, the 60S subunit, and guanine nucleotides. SBDS acts as a GDP exchange factor or GEF for EFL1, favouring the active GTP-bound conformation of the enzyme [ 5 , 6 ]. Based on low-resolution cryo-EM data, EFL1 binds the GTPase-associated centre of the 60S subunit and undergoes an SBDS-dependent conformational change, whereby its catalytic module (domains I and II) sterically displaces eIF6 [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Altered Conformational Landscape upon Sensing Guanine Nucleotides in a Disease Mutant of Elongation Factor-like 1 (EFL1) GTPase

et al. 2022

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The final maturation step of the 60S ribosomal subunit requires the release of eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) to enter the pool of mature ribosomes capable of engaging in translation. This process is mediated by the concerted action of the Elongation Factor-like 1 (human EFL1, yeast Efl1) GTPase and its effector, the Shwachman-Bodian-Diamond syndrome protein (human SBDS, yeast Sdo1). Mutations in these proteins prevent the release of eIF6 and cause a disease known as Shwachman–Diamond Syndrome (SDS). While some mutations in EFL1 or SBDS result in insufficient proteins to meet the cell production of mature large ribosomal subunits, others do not affect the expression levels with unclear molecular defects. We studied the functional consequences of one such mutation using Saccharomyces cerevisiae Efl1 R1086Q, equivalent to human EFL1 R1095Q described in SDS patients. We characterised the enzyme kinetics and energetic basis outlining the recognition of this mutant to guanine nucleotides and Sdo1, and their interplay in solution. From our data, we propose a model where the conformational change in Efl1 depends on a long-distance network of interactions that are disrupted in mutant R1086Q, whereby Sdo1 and the guanine nucleotides no longer elicit the conformational changes previously described in the wild-type protein. These findings point to the molecular malfunction of an EFL1 mutant and its possible impact on SDS pathology.

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Cooperative energetic effects elicited by the yeast Shwachman-Diamond syndrome protein (Sdo1) and guanine nucleotides modulate the complex conformational landscape of the elongation factor-like 1 (Efl1) GTPase

Cited by 8 publications

References 68 publications

Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman–Diamond Syndrome Subjects

Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman–Diamond Syndrome Subjects

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli

Altered Conformational Landscape upon Sensing Guanine Nucleotides in a Disease Mutant of Elongation Factor-like 1 (EFL1) GTPase

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