Brigitte A. van Oirschot

Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK†

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK‐deficient patients from 35 unrelated families. Twenty‐nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in‐frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N‐terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three‐dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme‐linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype‐to‐phenotype correlation in PK deficiency as well as the enzymes structure and function. Hum Mutat 0, 1–8, 2008.

Protein tyrosine phosphatase activity as a diagnostic parameter in breast cancer

SummaryCellular phosphotyrosine levels are regulated by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). It is supposed that this balance is disturbed in tumour cells, making the increased or altered activity of PTKs and PTPs likely hallmarks of tumour tissues. Indeed it could be shown that the PTK activity was increased in breast cancer in correlation with prognosis (Hennipmanet al., Cancer Res. 49, 516–522, 1989). In the present report we measured the PTP activities in breast cancer and normal breast tissues. An increase of approximately three- to four-fold was measured in the cytosolic tumour fractions compared to normal, whereas the solubilized membrane fraction PTP activity showed an increase in tumours of approximately 1.5-fold. Remarkably, the membrane PTP activity correlated with the presence of tumour positive axillary lymph nodes (p = 0.004), whereas the cytosolic PTP activity correlated with the mitotic index, a higher PTP activity occurring when the mitotic index was higher than 10 (p = 0.0004). These results indicate that membrane PTP activity may be considered as an index of metastatic potential, whereas cytosolic PTP activity may be a measure of the growth capacity of the tumour. The increase of PTP activity in breast cancers was confirmed by enzyme-histochemical studies. In frozen sections of tumours a strong to moderate activity was found in both tumour cells and interstitial cells. In the interstitium membrane activity was most pronounced, whereas in the tumour cells diffuse staining of the cytoplasm together with a clear membrane staining was demonstrated. Immunoblotting with anti-phosphotyrosine antibodies also reveals differences between the tumours and normal tissues, confirming the disturbance of the balance between protein tyrosyl phosphorylation and dephosphorylation in the tumour cells.

Taxol(®)-induced phosphatidylserine exposure and microvesicle formation in red blood cells is mediated by its vehicle Cremophor(®) EL.

AIM The conventional clinical formulation of paclitaxel (PTX), Taxol®, consists of Cremophor® EL (CrEL) and ethanol. CrEL-formulated PTX is associated with acute hypersensitivity reactions, anemia and cardiovascular events. In this study, the authors investigated the effects of CrEL-PTX on red blood cells (RBCs) and compared these with the effects observed after exposure to the novel nanoparticle albumin-bound PTX, marketed as Abraxane®. RESULTS The authors demonstrate that CrEL is primarily responsible for RBC lysis and induction of phosphatidylserine exposure. Phosphatidylserine-exposing RBCs showed increased association with endothelial cells in culture. The authors also identified CrEL as being responsible for vesiculation of RBCs. This is the first time that excipients have been shown to be involved in microvesicle formation. Microvesicles were taken up by endothelial cells. CONCLUSION These results offer new insights into the side effect profile of Taxol, which is likely to have implications for patients with erythrocyte disorders. Abraxane did not induce any of these effects on RBCs, indicating that the choice of excipients can have a pronounced influence on the efficacy and side effects of drug molecules.

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.

Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency. Am. J. Hematol. 90:E35–E39, 2015.

Novel Homozygous Mutation of the Internal Translation Initiation Start Site of VHL is Exclusively Associated with Erythrocytosis : Indications for Distinct Functional Roles of von Hippel-Lindau Tumor Suppressor Isoforms

Congenital secondary erythrocytosis is a rare disorder characterized by increased red blood cell production. An important cause involves defects in the oxygen sensing pathway, in particular the PHD2–VHL–HIF axis. Mutations in VHL are also associated with the von Hippel‐Lindau tumor predisposition syndrome. The differences in phenotypic expression of VHL mutations are poorly understood. We report on three patients with erythrocytosis, from two unrelated families. All patients show exceptionally high erythropoietin (EPO) levels, and are homozygous for a novel missense mutation in VHL: c.162G>C p.(Met54Ile). The c.162G>C mutation is the most upstream homozygous VHL mutation described so far in patients with erythrocytosis. It abolishes the internal translational start codon, which directs expression of VHLp19, resulting in the production of only VHLp30. The exceptionally high EPO levels and the absence of VHL‐associated tumors in the patients suggest that VHLp19 has a role for regulating EPO levels that VHLp30 does not have, whereas VHLp30 is really the tumor suppressor isoform.

Glucose 6-phosphate dehydrogenase deficiency in an elite long-distance runner.

To the editor: Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the worlds most common enzymopathy[1][1] and caused by mutations in the X-chromosomal G6PD gene. The enzyme catalyzes the first reaction of the hexose monophosphate shunt, which results in 2 main products: pentose phosphate

Proteomics reveals reduced expression of transketolase in pyrimidine 5′-nucleotidase deficient patients

To date, it remains a challenge to correctly and timely diagnose red blood cell (RBC) enzymopathies that result in hereditary nonspherocytic hemolytic anemia (HNSHA), the third most common of which is pyrimidine 5′‐nucleotidase (P5N) deficiency with just over 100 cases recognized and confirmed worldwide.

Molecular characterization of six new cases of red blood cell hexokinase deficiency yields four novel mutations in HK1.

Hexokinase (HK) is a key enzyme of glycolysis, the only metabolic pathway able to provide the red blood cell with ATP. HK deficiency is a very rare hereditary disorder with severe chronic nonspherocytic hemolytic anemia (HNSHA) as a major clinical feature. To date, only 24 patients with HK deficiency have been identified. Here, we report the molecular analysis of six new cases of HK deficiency. A total of six different mutations were detected in HK1, four of them described here for the first time: c.2599C>T p.(His867Tyr), c.1799C>T p.(Thr600Met), c.873-2A>G and c.493-1G>A. The pathogenic nature of the identified missense mutations was confirmed by biochemical and 3-dimensional structural analysis. The effects of the novel splice site mutation c.873-2A>G were studied at the level of pre-mRNA processing, and confirmed at the protein level. All together, these results provide a better insight into the pathogenesis of this rare red cell disorder, and contribute to a better understanding of the genotype-phenotype correlation in HK deficiency.