Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo

The role of N-myc downstream regulated gene-1 (NDRG1) in cancer has recently gained interest, as potential regulator of cell death and tumor suppressor. Although its normal function in the pancreas is largely unknown, loss of NDRG1 expression is associated with a more aggressive tumor phenotype and poor outcome in pancreatic cancer patients.

Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML

Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress-associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).

BIRC6 (APOLLON) is down-regulated in acute myeloid leukemia and its knockdown attenuates neutrophil differentiation

Background Inhibitors of apoptosis (IAPs) were intensively investigated in the context of cancer where they promote tumor growth and chemoresistence. Overexpression of the IAP BIRC6 is associated with unfavorable clinical features and negatively impacts relapse-free survival in childhood acute myeloid leukemia (AML). Currently, BIRC6 levels in adult primary AML have not been compared to the expression in normal myeloid cells. Thus, we compared for the first time BIRC6 levels in adult primary AML patient samples to normal myeloid cells and studied its regulation and function during neutrophil differentiation. Findings We found significantly lower BIRC6 levels in particular AML subtypes as compared to granulocytes from healthy donors. The lowest BIRC6 expression was found in CD34+ progenitor cells. Moreover, BIRC6 expression significantly increased during neutrophil differentiation of AML cell lines and knocking down BIRC6 in NB4 acute promyelocytic leukemia (APL) cells significantly impaired neutrophil differentiation, but not cell viability. Conclusion Together, we found an association of low BIRC6 levels with an immature myeloid phenotype and describe a function for BIRC6 in neutrophil differentiation of APL cells.

Agrin is required for survival and function of monocytic cells

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma

BACKGROUND ; AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes

BACKGROUND Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.

A Gly98Val mutation in the N-Myc downstream regulated gene 1 (NDRG1) in Alaskan Malamutes with polyneuropathy.

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

Shifts in microbial communities do not explain the response of grassland ecosystem function to plant functional composition and rainfall change

Ecosystem functioning in grasslands is regulated by a range of biotic and abiotic factors, and the role of microbial communities in regulating ecosystem function has been the subject of much recent scrutiny. However, there are still knowledge gaps regarding the impacts of rainfall and vegetation change upon microbial communities and the implications of these changes for ecosystem functioning. We investigated this issue using data from an experimental mesotrophic grassland study in south-east England, which had been subjected to four years of rainfall and plant functional composition manipulations. Soil respiration, nitrogen and phosphorus stocks were measured, and the abundance and community structure of soil microbes were characterised using quantitative PCR and multiplex-TRFLP analysis, respectively. Bacterial community structure was strongly related to the plant functional composition treatments, but not the rainfall treatment. However, there was a strong effect of both rainfall change and plant functional group upon bacterial abundance. There was also a weak interactive effect of the two treatments upon fungal community structure, although fungal abundance was not affected by either treatment. Next, we used a statistical approach to assess whether treatment effects on ecosystem function were regulated by the microbial community. Our results revealed that ecosystem function was influenced by the experimental treatments, but was not related to associated changes to the microbial community. Overall, these results indicate that changes in fungal and bacterial community structure and abundance play a relatively minor role in determining grassland ecosystem function responses to precipitation and plant functional composition change, and that direct effects on soil physical and chemical properties and upon plant and microbial physiology may play a more important role.

Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants

Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.