Impaired growth, hematopoietic colony formation, and ribosome maturation in human cells depleted of Shwachman-Diamond syndrome protein SBDS

2012 ◽  
Vol 60 (2) ◽  
pp. 281-286 ◽  
Author(s):  
Gulay Sezgin ◽  
Adrianna L. Henson ◽  
Abdallah Nihrane ◽  
Sharon Singh ◽  
Max Wattenberg ◽  
...  
Keyword(s):  
Colony Formation ◽  
Human Cells ◽  
Shwachman Diamond Syndrome ◽  
Syndrome Protein

Impaired Ribosome Maturation In Human Cells Depleted of Shwachman-Diamond Syndrome Protein SBDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2242-2242
Author(s):  
Gulay Sezgin ◽  
Abdallah Nihrane ◽  
Adrianna Henson ◽  
Max Wattenberg ◽  
Steven Ellis ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
A549 Cells ◽  
Autosomal Recessive Disorder ◽  
Human Cells ◽  
Yeast Cells ◽  
Cycle Phase ◽  
Shwachman Diamond Syndrome ◽  
Pancreatic Exocrine Dysfunction

Abstract Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

scholarly journals Shwachman-Diamond Syndrome Protein SBDS Maintains Human Telomeres by Regulating Telomerase Recruitment

Cell Reports ◽  
2018 ◽  
Vol 22 (7) ◽  
pp. 1849-1860 ◽  
Author(s):  
Yi Liu ◽  
Feng Liu ◽  
Yizhao Cao ◽  
Huimin Xu ◽  
Yangxiu Wu ◽  
...  
Keyword(s):  
Shwachman Diamond Syndrome ◽  
Syndrome Protein

Action-at-a-Distance Mutagenesis Induced by Oxidized Guanine in Werner Syndrome Protein-Reduced Human Cells

2015 ◽  
Vol 28 (4) ◽  
pp. 621-628 ◽  
Author(s):  
Hiroyuki Kamiya ◽  
Daiki Yamazaki ◽  
Eri Nakamura ◽  
Tetsuaki Makino ◽  
Miwako Kobayashi ◽  
...  
Keyword(s):  
Werner Syndrome ◽  
Human Cells ◽  
Werner Syndrome Protein ◽  
Action At A Distance ◽  
Oxidized Guanine ◽  
Syndrome Protein

Linking Defective Ribosome Maturation to Shwachman-Diamond Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-36-SCI-36
Author(s):  
Alan John Warren
Keyword(s):  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Ribosome Assembly ◽  
Genetic Dissection ◽  
Gtp Hydrolysis ◽  
Shwachman Diamond Syndrome ◽  
Novel Therapeutic Strategies ◽  
Syndrome Protein

Abstract Ribosomes are RNA-protein machines that translate the genetic information encoded by the mRNA template in all living cells. Recent high-resolution structures of the ribosome have revolutionized our understanding of protein translation. However, the mechanisms of ribosome assembly and the surveillance mechanisms that monitor this process and couple it to growth are poorly understood. Causative mutations and deletions of genes involved in ribosome biogenesis define an emerging group of disorders known as the ribosomopathies. Recent work from my laboratory strongly supports the hypothesis that Shwachman-Diamond syndrome (SDS) is a ribosomopathy caused by defective maturation of the large ribosomal subunit. Elucidation of the specific function of the SBDS protein that is deficient in SDS is revealing unexpected new insights that extend our understanding of the mechanisms underlying the late cytoplasmic steps of ribosome assembly and the quality control surveillance pathways that monitor 60S maturation. Genetic dissection of this pathway may inform novel therapeutic strategies for SDS. 1. Wong C.C., Traynor D., Basse N., Kay R.R., Warren A.J. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 2. Finch A.J., Hilcenko C., Basse N., Drynan L.F., Goyenechea B., Menne T.F., González Fernández Á., Simpson P., D’Santos C.S., Arends M.J., Donadieu J., Bellanné-Chantelot C., Costanzo M., Boone C., McKenzie A.N., Freund S.M., Warren A.J. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes and Development (2011) 25: 917-929. 3. Menne T.M., Goyenechea B., Sánchez-Puig N., Wong C.C., Tonkin L.M., Ancliff P., Brost R.L., Costanzo M., Boone C. and Warren A.J. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interaction of the GTPase Elongation Factor Like-1 with the Shwachman-Diamond Syndrome Protein and Its Missense Mutations

2018 ◽  
Vol 19 (12) ◽  
pp. 4012 ◽  
Author(s):  
Abril Gijsbers ◽  
Diana Montagut ◽  
Alfonso Méndez-Godoy ◽  
Davide Altamura ◽  
Michele Saviano ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Interaction Mechanism ◽  
Elongation Factor ◽  
Missense Mutations ◽  
Molecular Alterations ◽  
X Ray Scattering ◽  
Genes Encoding ◽  
Initial Interaction ◽  
Shwachman Diamond Syndrome ◽  
Syndrome Protein

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2–3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.

scholarly journals The Activated TGFβ Pathway in Shwachman Diamond Syndrome Impairs Hematopoiesis and Is Down-Regulated By Deletion of 7q

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 875-875
Author(s):  
Melisa Ruiz-Gutierrez ◽  
Cailin Joyce ◽  
Ozge Vargel Bolukbasi ◽  
Andriana Kotini ◽  
Gabriela Alexe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Failure ◽  
Myeloid Differentiation ◽  
Monosomy 7 ◽  
Relative Growth ◽  
Cd34 Cells ◽  
Shwachman Diamond Syndrome ◽  
Tgfβ Pathway ◽  
Normal Controls

Abstract Monosomy 7 or deletion of 7q (del(7q)) are common cytogenetic abnormalities in pediatric MDS. Monosomy 7/del(7q) frequently arises in the context of inherited bone marrow failure (BMF) syndromes such as Shwachman Diamond Syndrome (SDS), an autosomal recessive disorder caused by biallelic mutations in the SBDS gene. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited BMF. The basis for this propensity to develop monosomy 7/del(7q) remains unclear. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in BMF, remains a critical question. The aim of this study is to investigate the molecular consequences of del(7q) in the context of BMF with the goal of developing more effective treatments. The lack of synteny between murine and human chromosome 7 has posed a major barrier to modeling monosomy 7/del(7q). Therefore, we utilized SDS patient-derived induced pluripotent stem cells (SDS-iPSC), which recapitulate the hematopoietic defects of this BMF syndrome. Independent SDS-iPSC lines were derived from two patients with SDS. Proliferationof SDS-iPSCs was reduced relative to wild type controls without a concomitant increase in cell death. Compared to normal iPSC controls, SDS iPSC-derived CD34+ cells exhibited reduced hematopoietic differentiation to CD45+ cells, decreased erythroid and myeloid hematopoietic progenitor colony formation in methylcellulose, and impaired terminal myeloid differentiation. To study the molecular and biologic effects of del(7q), a deletion of the MDS-associated region of the long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q in SDS iPSCs spanned band q11.23 to q36.3. The SDS-del(7q) iPSCs expressed stem cell markers and formed teratomas as efficiently as their isogenic SDS iPSC controls.Deletion of 7q failed to confer a relative growth advantage even within the context of BMF. Indeed, proliferation of the SDS-del(7q) iPSCs was reduced below that of both the isogenic SDS iPSCs and normal controls without an increase in cell death. To investigate the effect of del(7q) on hematopoiesis, iPSC-derived CD34+ cells were assayed for their ability to undergo hematopoietic differentiation. SDS-del(7q) CD34+ cells demonstrated reduced differentiation to CD45+ cells, reduced methylcellulose hematopoietic progenitor colony formation and impaired terminal myeloid differentiation compared with isogenic SDS CD34+ cells. These data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic SDS iPSCs and results in a further impairment of hematopoiesis. To gain insight into the mechanisms of del7q-associated clonal evolution in SDS, we performed RNA sequencing (RNAseq) of SDS and SDS-del(7q) iPSC. Expression of TGFβ pathways and their downstream targets were reduced in SDS del(7q) iPSCs compared to isogenic SDS iPSC controls. This observation was intriguing because single cell RNAseq analysis of primary SDS bone marrow cells revealed that TGFβ pathway is hyperactivated in SDS. Additionally, a quantitative DNA aptamer-based proteomic analysis demonstrated elevated levels of TGFβ ligands in plasma from six SDS patients compared to 6 age-matched controls. Western blot analysis showed increased P-SMAD2 levels in SDS iPSCs compared to SDS-del(7q) and normal controls, while total levels of SMAD2 were unchanged. Pharmacological targeting of TFGβ with small molecule inhibitors resulted in selective improvement of SDS hematopoietic colony formation without stimulating outgrowth of the isogenic SDS-del(7q) cells. Although del(7q) does not seem to confer an advantage in growth or hematopoiesis in vitro, we are testing whether the deletion of 7q might confer relative resistance to elevated TGFβ levels in vivo, resulting in an overall survival advantage of the del7q clone.These results demonstrate that the TGFβ pathway is activated in SDS, and that deletion of 7q reverses the TGFβ pathway hyperactivation in SDS. Inhibition of TGFβ selectively rescues hematopoiesis in SDS but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. Disclosures Stegmaier: Novartis: Consultancy, Research Funding. Shimamura: TransCellular Therapeutics: Other: Spouse is majority shareholder.

scholarly journals Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

2016 ◽  
Vol 7 (3) ◽  
pp. 187-200 ◽  
Author(s):  
Chengying Ma ◽  
Kaige Yan ◽  
Dan Tan ◽  
Ningning Li ◽  
Yixiao Zhang ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Shwachman Diamond Syndrome ◽  
Syndrome Protein

scholarly journals Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells

2018 ◽  
Vol 93 (4) ◽  
pp. 527-536 ◽  
Author(s):  
Valentino Bezzerri ◽  
Donatella Bardelli ◽  
Jacopo Morini ◽  
Antonio Vella ◽  
Simone Cesaro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Function ◽  
Bone Marrow Cells ◽  
Shwachman Diamond Syndrome ◽  
Syndrome Protein ◽  
Marrow Cells
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