Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein.

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.

Novel Bispecific Antibody for Synovial-Specific Target Delivery of Anti-TNF Therapy in Rheumatoid Arthritis.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

Gut-Ex-Vivo system as a model to study gluten response in celiac disease.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

Defining the Helicobacter pylori Disease-Specific Antigenic Repertoire.

The analysis of the interaction between Helicobacter pylori (HP) and the host in vivo is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific "disease signature" with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines "phage display" and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen's genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future.

SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease.

Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD.

Aldo-keto reductases protect metastatic melanoma from ER stress-independent ferroptosis.

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.

Antisense Transcription in Loci Associated to Hereditary Neurodegenerative Diseases.

Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.

Ecto-Calreticulin is essential for an efficient immunogenic cell death stimulation in mouse melanoma.

Skin melanoma remains one of the most aggressive and difficult to treat human malignancy, with an increasing incidence every year. Although surgical resection represents the best therapeutic approach, this is only feasible in cases of early diagnosis. Furthermore, the established malignancy is resistant to all therapeutic strategies employed so far, resulting in an unacceptable patient survival rate. Although the immune-mediated therapeutic approaches, based on anti-PD1 or anti-CTLA4, are very promising and under clinical trial experimentation, they could conceal not yet fully emerged pitfalls such as the development of autoimmune diseases. Therefore, alternative therapeutic approaches are still under investigation, such as the immunogenic cell death (ICD) process. Here we show that the lack of calreticulin translocation onto mouse melanoma cell membrane prevents the stimulation of an effective ICD response in vivo.

Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.

The interaction between the enzyme transglutaminase 2 (TG2) and fibronectin (FN) is involved in the cell-matrix interactions that regulate cell signaling, adhesion, and migration and play central roles in pathologic conditions, particularly fibrosis and cancer. A precise definition of the exact interaction domains on both proteins could provide a tool to design novel molecules with potential therapeutic applications. Although specific residues involved in the interaction within TG2 have been analyzed, little is known regarding the TG2 binding site on FN. This site has been mapped to a large internal 45-kDa protein fragment coincident with the gelatin binding domain (GBD). With the goal of defining the minimal FN interacting domain for TG2, we produced several expression constructs encoding different portions or modules of the GBD and tested their binding and functional properties. The results demonstrate that the I8 module is necessary and sufficient for TG2-binding in vitro, but does not have functional effects on TG2-expressing cells. Modules I7 and I9 increase the strength of the binding and are required for cell adhesion. A 15-kDa fragment encompassing modules I7-9 behaves as the whole 45-kDa GBD and mediates signaling, adhesion, spreading, and migration of TG2+ cells. This study provides new insights into the mechanism for TG2 binding to FN.-Soluri, M. F., Boccafoschi, F., Cotella, D., Moro, L., Forestieri, G., Autiero, I., Cavallo, L., Oliva, R., Griffin, M., Wang, Z., Santoro, C., Sblattero, D. Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.

PKR and GCN2 stress kinases promote an ER stress-independent eIF2α phosphorylation responsible for calreticulin exposure in melanoma cells.

The immunogenic cell death (ICD) process represents a novel therapeutic approach to treat tumours, in which cytotoxic compounds promote both cancer cell death and the emission of damage-associated molecular patterns (DAMPs) from dying cells, to activate the immune system against the malignancy. Therefore, we explored the possibility to stimulate the key molecular players with a pivotal role in the execution of the ICD program in melanoma cells. To this aim, we used the pro-ICD agents mitoxantrone and doxorubicin and found that both agents could induce cell death and stimulate the release/exposure of the strictly required DAMPs in melanoma cells: i) calreticulin (CRT) exposure on the cell membrane; ii) ATP secretion; iii) type I IFNs gene up-regulation and iv) HMGB1 secretion, highlighting no interference by oncogenic BRAF. Importantly, although the ER stress-related PERK activation has been linked to CRT externalization, through the phosphorylation of eIF2α, we found that this stress pathway together with PERK were not involved in melanoma cells. Notably, we identified PKR and GCN2 as key mediators of eIF2α phosphorylation, facilitating the translocation of CTR on melanoma cells surface, under pro-ICD drugs stimulation. Therefore, our data indicate that pro-ICD drugs are able to stimulate the production/release of DAMPs in melanoma cells at least in vitro, indicating in this approach a potential new valuable therapeutic strategy to treat human skin melanoma malignancy.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.

Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia.

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with huntingtin protein and promotes its atypical ubiquitination to enhance aggregate formation.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

The 423Q polymorphism of the X-linked inhibitor of apoptosis gene influences monocyte function and is associated with periodic fever.

OBJECTIVE: Hereditary periodic fever syndromes (HPFs) develop as a result of uncontrolled activation of the inflammatory response, with a substantial contribution from interleukin-1beta or tumor necrosis factor alpha (TNFalpha). The HPFs include familial Mediterranean fever (FMF), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), TNF receptor-associated syndrome (TRAPS), and cryopyrinopathies, which are attributable to mutations of the MEFV, MVK, TNFRSF1A, and CIAS1 genes, respectively. However, in many patients, the mutated gene has not been determined; therefore, the condition in these patients with an HPF-like clinical picture is referred to as idiopathic periodic fever (IPF). The aim of this study was to assess involvement of X-linked inhibitor of apoptosis (XIAP), which plays a role in caspase inhibition and NF-kappaB signaling, both of which are processes that influence the development of inflammatory cells. METHODS: The XIAP gene (X-linked) was sequenced in 87 patients with IPF, 46 patients with HPF (13 with HIDS, 17 with TRAPS, and 16 with FMF), and 182 healthy control subjects. The expression of different alleles was evaluated by sequencing XIAP-specific complementary DNA mini-libraries and by real-time polymerase chain reaction and Western blot analyses. The functional effect of XIAP on caspase 9 activity was assessed by a fluorimetric assay, and cytokine secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: Sequencing disclosed a 1268A>C variation that caused a Q423P amino acid substitution. The frequency of 423Q-homozygous female patients and 423Q-hemizygous male patients was significantly higher in the IPF group than in the control group (69% versus 51%; odds ratio 2.17, 95% confidence interval 1.23-3.87, P = 0.007), whereas no significant difference was detected in the HPF group (59%) compared with controls. In primary lymphocytes and transfected cell lines, 423Q, as compared with 423P, was associated with higher XIAP protein and messenger RNA expression and lower caspase 9 activation. In lipopolysaccharide-activated monocytes, 423Q was associated with higher secretion of TNFalpha. CONCLUSION: These results suggest that 423Q is a predisposing factor for IPF development, possibly through its influence on monocyte function.

Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer.

BACKGROUND: Diamond-Blackfan anaemia (DBA) is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. RESULTS: To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. CONCLUSION: This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis.

Activation of caspase-8 triggers anoikis in human neuroblastoma cells.

Cells require appropriate interaction with extracellular matrix proteins mediated by integrins to grow, differentiate and survive. Many cell types including nervous cells undergo anoikis, a substrate-dependent apoptosis, when adhesion is impaired. Resistance of tumors to cytotoxic drugs is probably due to disturbed apoptosis programs. The proteolytic enzymes caspases are the main executioners of apoptosis. It was reported that caspase-8 expression is deficient in some neuroblastoma cells. We demonstrated that human neuroblastoma cell line SK-B-BE, differentiated with retinoic acid, expressed caspases 3, 8 and 9. Caspases 8 and 3, but not caspase-9 were activated in SK-N-BE cells cultured in suspension or on aspecific adhesive substrate. Cell positive to caspase-8 were classified into four stages, by morphometric and densitometric parameters. The use of the specific caspase-8 inhibitor Z-IETD-FMK dramatically reduced apoptosis, demonstrating that caspase-8 is the upstream initiator caspase during SK-N-BE cells anoikis. Among matrix proteins, type I collagen is the most effective and fibronectin the least in delaying anoikis. The activation of caspases 8 and 3 by unligated integrins was dependent on the state of neuronal differentiation, since the most differentiated cell was the most vulnerable to anoikis. These data show that activation of caspase-8 is specifically required to promote anoikis in SK-N-BE neuroblastoma cells.

Interactions between RPS19, mutated in Diamond-Blackfan anemia, and the PIM-1 oncoprotein.

BACKGROUND AND OBJECTIVES: Diamond Blackfan anemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation. Patients' bone marrow progenitor cells do not respond to erythropoietic growth factors, such as erythropoietin. Mutations in the gene encoding for ribosomal protein (RP) S19 account for 25% of cases of DBA. The link between defective erythropoiesis and RPS19 is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown extraribosomal functions. DESIGN AND METHODS: We used yeast two-hybrid screening and a human liver cDNA library obtained at 19-24 weeks of gestation, when hepatic erythropoiesis is efficient, to search for proteins interacting with RPS19. RESULTS: We found that RPS19 binds PIM-1, an ubiquitous serine-threonine kinase whose expression can be induced in erythropoietic cells by several growth factors, such as erythropoietin. The PIM-1/RPS19 interaction was demonstrated both in vitro and in living cells and led to phosphorylation of RPS19 in an in vitro kinase assay. We also showed that in human 293T cells PIM-1 interacts with ribosomes and may be involved in translational control. Three DBA-associated RPS19 mutations alter the binding between RPS19 and PIM-1. INTERPRETATION AND CONCLUSIONS: A link between erythropoietic growth factor signaling and RPS19 has been identified for the first time.

Modification of the erythroid transcription factor GATA-1 by SUMO-1.

The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.