scholarly journals Impaired ribosomal subunit association in Shwachman-Diamond syndrome

Blood ◽  
2012 ◽  
Vol 120 (26) ◽  
pp. 5143-5152 ◽  
Author(s):  
Nicholas Burwick ◽  
Scott A. Coats ◽  
Tomoka Nakamura ◽  
Akiko Shimamura

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive marrow failure syndrome with a predisposition to leukemia. SDS patients harbor biallelic mutations in the SBDS gene, resulting in low levels of SBDS protein. Data from nonhuman models demonstrate that the SBDS protein facilitates the release of eIF6, a factor that prevents ribosome joining. The complete abrogation of Sbds expression in these models results in severe cellular and lethal physiologic abnormalities that differ from the human disease phenotype. Because human SDS cells are characterized by partial rather than complete loss of SBDS expression, we interrogated SDS patient cells for defects in ribosomal assembly. SDS patient cells exhibit altered ribosomal profiles and impaired association of the 40S and 60S subunits. Introduction of a wild-type SBDS cDNA into SDS patient cells corrected the ribosomal association defect, while patient-derived SBDS point mutants only partially improved subunit association. Knockdown of eIF6 expression improved ribosomal subunit association but did not correct the hematopoietic defect of SBDS-deficient cells. In summary, we demonstrate an SBDS-dependent ribosome maturation defect in SDS patient cells. The role of ribosomal subunit joining in marrow failure warrants further investigation.

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3438-3438
Author(s):  
Nicholas Burwick ◽  
Scott Coats ◽  
Akiko Shimamura

Abstract Abstract 3438 Shwachman Diamond syndrome (SDS) is an autosomal recessive marrow failure syndrome with a predisposition to leukemia. Over 90% of SDS patients harbor biallelic mutations in the SBDS gene. SBDS has been implicated in several cellular functions including ribosome biogenesis and mitotic spindle stabilization. Deletion of SBDS orthologues in yeast results in a severe slow growth phenotype and depressed polysomes. Homozygous deletion of Sbds in murine models results in early embryonic lethality, while conditional deletion of Sbds in mouse liver demonstrates accumulation of 40S and 60S subunits and halfmer formation consistent with impaired ribosome joining. SBDS facilitates the release of eIF6, a factor that prevents ribosome joining. The dramatic phenotypic and polysome changes noted in these experimental models were not observed in cells derived from SDS patients. SDS patient cells have only a mildly reduced growth rate compared to heatlhy controls, and polysome profiles do not demonstrate depressed polysomes or halfmer formation. Since complete abrogation of SBDS expression is lethal and biallelic null mutations in SBDS have not been reported, we examined the role of SBDS and eIF6 in SDS patients and human cell models. We first investigated whether ribosome subunit homeostasis is impaired in SDS patient cells. We find that the 60S:40S ribosomal subunit ratio is consistently reduced in bone marrow stromal cells from SDS patients of different genotypes (n=4). This impairment in 60S:40S ratio is demonstrated in both SDS patient stromal cells and patient lymphoblasts. Stable lentiviral knockdown of SDS in normal marrow stromal cells recapitulates the reduction in 60S:40S ratio. SBDS and eIF6 co-sediment in polysome gradients of human SDS cells. This co-sedimentation is specific for the 60S ribosomal subunit. Since eIF6 has a role as an anti-joining factor, we next developed an in vitro assay to test for ribosome subunit joining in human cells. In this assay, we validate that over-expression of eIF6 results in reduced ribosome joining, and eIF6 knockdown promotes ribosome joining. Moreover, we find that SDS patient stromal cells and patient lymphoblasts both demonstrate impaired ribosome subunit joining, compared with healthy controls. Importantly, the addition of wild type SBDS or depletion of eIF6 improve ribosome joining in SDS patient cells. We demonstrate that the amino terminal sequences of SBDS are necessary but not sufficient for the association of SBDS with the 60S ribosomal subunit. Insertion of a patient-derived N-terminal SBDS point mutation also results in decreased association of SBDS with the 60S ribosomal subunit. These structure-function studies may help to inform genotype:phenotype correlations in SDS. The role of defective ribosome joining in promoting the SDS hematopoietic phenotype is of particular interest. Ongoing studies are interrogating the role of eIF6 modulation on the hematopoietic phenotype in SBDS- depleted cells. Insights garnered from these experiments will help inform the development of novel agents to improve the hematopoetic defect in human SDS. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2019 ◽  
Vol 134 (3) ◽  
pp. 277-290 ◽  
Author(s):  
Shengjiang Tan ◽  
Laëtitia Kermasson ◽  
Angela Hoslin ◽  
Pekka Jaako ◽  
Alexandre Faille ◽  
...  

Abstract Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.


2021 ◽  
Vol 22 (14) ◽  
pp. 7360
Author(s):  
Angie De La Cruz ◽  
Aubrey Hargrave ◽  
Sri Magadi ◽  
Justin A. Courson ◽  
Paul T. Landry ◽  
...  

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the β2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.


2007 ◽  
Vol 292 (3) ◽  
pp. H1435-H1442 ◽  
Author(s):  
Taiji Yamaguchi ◽  
Kazuhiro Kamada ◽  
Catherine Dayton ◽  
F. Spencer Gaskin ◽  
Mozow Yusof ◽  
...  

Ingestion of low levels of ethanol 24 h before [ethanol preconditioning (EPC)] ischemia and reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and adhesion (LA), effects that were abolished by adenosine A2 receptor (ADO-A2R) antagonists or nitric oxide (NO) synthase (NOS) inhibitors. The aims of this study were to determine whether NO derived from endothelial NOS (eNOS) during the period of ethanol exposure triggered entrance into this preconditioned state and whether these events were initiated by an ADO-A2R-dependent mechanism. Ethanol or distilled water vehicle was administered to C57BL/6J [wild type (WT)] or eNOS-deficient (eNOS−/−) mice by gavage. Twenty-four hours later, the superior mesenteric artery was occluded for 45 min. LR and LA were quantified by intravital microscopy after 30 and 60 min of reperfusion. I/R increased LR and LA in WT mice, effects that were abolished by EPC or NO donor preconditioning (NO-PC). NO-PC was not attenuated by coincident administration of an ADO-A2R antagonist. I/R increased LR and LA in eNOS−/− mice to levels comparable with those noted in WT animals. However, EPC only slightly attenuated postischemic LR and LA, whereas NO-PC remained effective as a preconditioning stimulus in eNOS−/− mice. Preconditioning with an ADO-A2R agonist (which we previously demonstrated prevents I/R-induced LR and LA in WT animals) failed to attenuate these postischemic adhesive responses in eNOS−/− mice. Our results indicate that EPC is triggered by NO formed secondary to ADO-A2R-dependent eNOS activation during the period of ethanol exposure 24 h before I/R.


HortScience ◽  
2007 ◽  
Vol 42 (2) ◽  
pp. 403-406 ◽  
Author(s):  
Antal Szőke ◽  
Erzsébet Kiss ◽  
László Heszky ◽  
Ildikó Kerepesi ◽  
Ottó Toldi

The aim of this work was to examine the role of fructose 2,6-bisphosphate (fru 2,6P2) in the carbohydrate metabolism in carnation (Dianthus caryophyllus L.). For this purpose, transgenic plants harboring two modified bifunctional enzyme complementary DNAs of rat liver origin (6-phosphofructo-2-kinase/fructose 2,6-biphosphatase) were generated. Transformation with the kinase construct resulted in a 45% to 85% increase in fru 2,6P2 concentrations compared with the wild type. Transformation with the phosphatase construct reduced the fru 2,6P2 contents by 45% and 70%. These alterations in fru 2,6P2 amounts affected the key enzyme activities of sucrose and starch metabolism. Accordingly, plants with elevated fru 2,6P2 concentrations had high levels of starch, fructose, and triose phosphates, and low levels of sucrose, glucose, and hexose phosphates. In plants with reduced amounts of fru 2,6P2 different results could be observed in major carbohydrate compounds.


Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1336-1341 ◽  
Author(s):  
Stefano Duga ◽  
Rosanna Asselta ◽  
Elena Santagostino ◽  
Sirous Zeinali ◽  
Tatjana Simonic ◽  
...  

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen A-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bβ-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.


Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1980-1988 ◽  
Author(s):  
Zhonghua Zhang ◽  
Norma E. Ramirez ◽  
Thomas E. Yankeelov ◽  
Zhengzhi Li ◽  
Laura E. Ford ◽  
...  

To define the role of the α2β1 integrin in pathologic angiogenesis, we investigated tumor-associated growth and angiogenesis in wild-type and α2-null mice. Our findings reveal that the α2β1 integrin plays an important role in angiogenesis via regulation of VEGFR1 expression. When challenged with B16F10 melanoma cells, mice lacking α2β1 integrin ex-pression exhibit increased tumor angiogenesis associated with up-regulated VEGFR1 expression. In contrast, there was no α2β1 integrin-dependent difference in the angiogenic response to Lewis lung carcinoma (LLC) cells. Interestingly, whereas B16F10 cells secrete high levels of placental growth factor (PLGF), LLC cells produce high levels of VEGF, but low levels of PLGF. The α2β1 integrin-dependent difference in angiogenesis was restored to LLC cells by expression of PLGF, strongly suggesting that the angiogenic phenotype and tumor growth in the α2-null host is dependent on specific interactions between the tumor cell and the genetically defined integrin repertoire of the host microenvironment. Thus integrin α2-null mice represent an example of genetic alterations of “the soil” determining response to the “seed.”


Genetics ◽  
1980 ◽  
Vol 95 (3) ◽  
pp. 661-672
Author(s):  
Barry Yedvobnick ◽  
Hallie M Krider ◽  
Bryan I Levine

ABSTRACT The autosomal recessive, maternal-effect mutation abnormal oocyte (abo: 2-38) preferentially lowers the viability of XO progeny. The severity of the sex-ratio distortion is reduced by duplications of maternal or zygotic heterochromatin chromatin (SANDLER 1970, 1977; PARRY and SANDLER 1974). Utilizing X-chromosome inversions that contain modifications in the quantity and arrangement of the heterochromatic functions, Xhabo and cr  +, we have extended our investigations of nbo's influence on XO male recovery and rDNA redundancy (KRIDER,YEDVOBNICK and LEVINE 1979).——XO males bearing In(1)scs1Lsc4R or In(1)wm4Lsc4R are recovered twice as frequently as X chromosomes containing a single Xh region, implying that these inversions possess a duplication of Xhabo. abo mutant females heterozygous for In(1)scs1Lsc4R and wild-type X chromosomes generate XO progeny that do not contain elevated rDNA redundancies. XO males containing In(1)wm4 exhibit male recoveries and rDNA elevations similar to those of males bearing a wild-type X chromosome, when both derive from a common abo/abo mother. Reciprocal crosses between In(1)wm4 and Canton-S males to attached-X abo females show significant, though reduced, sex ratios in the absence of an rDNA effect. The observation that abo can elevate the rDNA redundancy of In(1)wm4, a chromosome that does not compensate, suggests that abo and cr+ functions are not directly related.


1998 ◽  
Vol 66 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Christopher Elkins ◽  
Pat A. Totten ◽  
Bonnie Olsen ◽  
Christopher E. Thomas

ABSTRACT By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressinghgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor inE. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes wasexbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.


Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 876-876
Author(s):  
Pekka Jaako ◽  
Chi C Wong ◽  
David Adams ◽  
Alan J. Warren

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and a striking propensity to develop poor prognosis myelodysplastic syndrome and acute myeloid leukemia. In 90 % of cases the disease is caused by biallelic mutations in the gene encoding SBDS. We have shown previously that SBDS is a cytoplasmic ribosome assembly factor that catalyzes the release of the eukaryotic initiation factor 6 (eIF6) from the subunit joining interface of 60S ribosomal subunit (Menne et al, 2007; Finch et al, 2011). Deficiency of SBDS therefore results in aberrant retention of eIF6 on the 60S subunits that in turn perturbs ribosomal subunit joining and the formation of translation-competent 80S ribosomes. However, the mechanism linking defective ribosome assembly to marrow failure and leukemia in SDS remain poorly understood. Lack of viable mouse models presents a barrier to progress in understanding SDS disease pathophysiology and to evaluate novel therapies. We hypothesized that induced overexpression of eIF6 would mimic the consequences of SBDS deficiency by reducing the cytoplasmic pool of free 60S subunits and impairing translation. To test this hypothesis we have generated a novel transgenic eIF6 mouse model for SDS using KH2 embryonic stem cells that constitutively express the M2-reverse tetracycline transactivator at the Rosa26 locus with the EIF6 gene targeted downstream of the Col1a1 locus. This strategy permits systemic doxycycline-inducible and graded overexpression of eIF6 through control of the transgene copy number. We have validated that eIF6 overexpression promotes an increase in eIF6-bound cytoplasmic 60S subunits with a concomitant reduction in 80S ribosomes and polysomes in c-kit+ hematopoietic progenitor cells isolated from the transgenic eIF6 mice, thereby recapitulating the ribosomal subunit joining defect observed in patients with SDS. In vitro, the hematopoietic progenitor cells exhibit a strict eIF6 dose-dependent expansion defect. In vivo, mice with graded eIF6 overexpression are viable but develop macrocytic anemia with reticulocytopenia, thrombocytosis and mild leukopenia. Bone marrow transfer experiments demonstrate that the phenotype is autonomous to the hematopoietic system. Longitudinal phenotypic analyses in primary and transplanted animals are ongoing. Flow cytometric analysis of the bone marrow from transgenic eIF6 mice reveals a significant increase in the frequencies of preCFU-E and CFU-E erythroid progenitor cells and erythroblasts, but a significant reduction in the frequency of reticulocytes. Furthermore, we observe a striking accumulation of abnormal orthochromatic erythroblast-like cells that appear to have failed to enucleate, comprising approximately 1.5 % of the total bone marrow cells. Amnis ImageStream analysis, which combines flow cytometry with fluorescent microscopy, reveals a significant decrease in the frequency of erythroblasts that are able to complete the enucleation process. To address the underlying mechanism, we hypothesized that by impairing the formation of translation-competent 80S ribosomes, eIF6 overexpression would reduce the global rate of protein synthesis. Indeed, O-propargyl-puromycin incorporation assays established that the erythroblasts from the transgenic eIF6 mice have an approximately 3-fold reduction in global protein synthesis rate. Furthermore, our preliminary data suggest that the erythroid phenotype is p53-independent. Finally, erythroblasts from the transgenic eIF6 mice show a significant increase in levels of reactive oxygen species, but the functional significance of this finding remains unclear. We conclude that reduced rates of global translation drive defective hematopoiesis in the transgenic eIF6 mice. Importantly, eIF6 overexpression in vivo phenocopies SBDS depletion in human CD34+ cells (Sen et al, 2011). Together with the recent discovery of DNAJC21 (the human homologue of the 60S ribosomal assembly factor JJJ1 in yeast) as an SDS disease gene, our data support the hypothesis that deregulated cytoplasmic 60S subunit maturation and reduced translation are the primary drivers of the hematopoietic defect in SDS. Our viable transgenic eIF6 mouse model provides a unique tool to further dissect the mechanisms that underlie bone marrow failure and malignant transformation in SDS and for the development of novel therapeutics. Disclosures No relevant conflicts of interest to declare.


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