RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling.     

Reaction kinetics in the plasma membrane

A great puzzle in science is establishing a bottom up understanding of life by revealing how a collection of molecules gives rise to a living cell that can survive, communicate, and reproduce. In the confines of physics, chemistry, or material science laboratories where it possible to study complex interactions between molecules in a well-defined environment, our understanding of collective behavior is substantially developed. However, the environment in which molecules of a biological cell perform their functions is far from ideal or controllable. The environment inside cellular regions such as the plasma membrane is heterogeneous and dynamic, and functional molecules such as proteins are both dynamic and promiscuous, as they interact with countless other molecules. This makes it extremely challenging to grasp the inner mechanism of the cells, both experimentally and theoretically. On the bright side, this presents scientists with a colorful playground that waits to be explored: the mesoscopic world inside the cell. This review covers some of the recent experimental and theoretical developments in the study of molecular interactions in the plasma membrane, viewed as a heterogeneous medium where the number of reactants can be small, sometimes countable, and its implications for biological function.     

Online nanoliquid chromatography-mass spectrometry and nanofluorescence detection for high-resolution quantitative N-glycan analysis

The characterization of the repertoire of glycans at the quantitative and qualitative levels on cells and glycoproteins is a necessary step to the understanding of glycan functions in biology. In addition, there is an increasing demand in the field of biotechnology for the monitoring of glycosylation of recombinant glycoproteins, an important issue with regard to their safety and biological activity. The enzymatic release followed by fluorescent derivatization of glycans and separation by normal phase high-performance liquid chromatography (HPLC) has proven for many years to be a powerful approach to the quantification of glycans. Characterization of glycans has classically been performed by mass spectrometry (MS) with external standardization. Here, we report a new method for the simultaneous quantification and characterization of the N-glycans on glycoproteins without the need for external standardization. This method, which we call glycan nanoprofiling, uses nanoLC-coupled electrospray ionization (ESI)-MS with an intercalated nanofluorescence reader and provides effective single glycan separation with subpicomolar sensitivity. The method relies on the isolation and coumaric derivatization of enzymatically released glycans collected by solid phase extraction with porous graphitized carbon and their separation over polyamide-based nanoHPLC prior to serial nanofluorescence and nanoelectrospray mass spectrometric analysis. Glycan nanoprofiling is a broadly applicable and powerful approach that is sufficient to identify and quantify many glycan oligomers in a single run. Glycan nanoprofiling was successfully applied to resolve the glycans of monoclonal antibodies, showing that this method is a fast and sensitive alternative to available methods.     

An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense.     

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.     

Mutations of ESRRB encoding estrogen-related receptor beta cause autosomal-recessive nonsyndromic hearing impairment DFNB35

In a large consanguineous family of Turkish origin, genome-wide homozygosity mapping revealed a locus for recessive nonsyndromic hearing impairment on chromosome 14q24.3-q34.12. Fine mapping with microsatellite markers defined the critical linkage interval to a 18.7 cM region flanked by markers D14S53 and D14S1015. This region partially overlapped with the DFNB35 locus. Mutation analysis of ESRRB, a candidate gene in the overlapping region, revealed a homozygous 7 bp duplication in exon 8 in all affected individuals. This duplication results in a frame shift and premature stop codon. Sequence analysis of the ESRRB gene in the affected individuals of the original DFNB35 family and in three other DFNB35-linked consanguineous families from Pakistan revealed four missense mutations. ESRRB encodes the estrogen-related receptor beta protein, and one of the substitutions (p.A110V) is located in the DNA-binding domain of ESRRB, whereas the other three are substitutions (p.L320P, p.V342L, and p.L347P) located within the ligand-binding domain. Molecular modeling of this nuclear receptor showed that the missense mutations are likely to affect the structure and stability of these domains. RNA in situ hybridization in mice revealed that Esrrb is expressed during inner-ear development, whereas immunohistochemical analysis showed that ESRRB is present postnatally in the cochlea. Our data indicate that ESRRB is essential for inner-ear development and function. To our knowledge, this is the first report of pathogenic mutations of an estrogen-related receptor gene.     

Human T Cell Activation Results in Extracellular Signal-regulated Kinase (ERK)-Calcineurin-dependent Exposure of Tn Antigen on the Cell Surface and Binding of the Macrophage Galactose-type Lectin (MGL)

The C-type lectin macrophage galactose-type lectin (MGL) exerts an immunosuppressive role reflected by its interaction with terminal GalNAc moieties, such as the Tn antigen, on CD45 of effector T cells, thereby down-regulating T cell receptor signaling, cytokine responses, and induction of T cell death. Here, we provide evidence for the pathways that control the specific expression of GalNAc moieties on human CD4⁺ T cells. GalNAc epitopes were readily detectable on the cell surface after T cell activation and required de novo protein synthesis. Expression of GalNAc-containing MGL ligands was completely dependent on PKC and did not involve NF-κB. Instead, activation of the downstream ERK MAPK pathway led to decreased mRNA levels and activity of the core 1 β3GalT enzyme and its chaperone Cosmc, favoring the expression of Tn antigen. In conclusion, expression of GalNAc moieties mirrors the T cell activation status, and thus only highly stimulated T cells are prone to the suppressive action of MGL.     

Investigation of CYP21A2 mutations in Turkish patients with 21-hydroxylase deficiency and a novel founder mutation

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessively inherited disorders characterized by impaired production of adrenal steroids. Approximately 95% of all CAH are caused by mutations of the CYP21A2 that encodes 21-hydroxylase. In this study, mutation analyses of CYP21A2 were performed in 48 CAH patients from 45 Turkish families with the clinical diagnosis of 21-hydroxylase deficiency (21OHD). While in 39 (86.7%) of 21OHD patients, disease causing CYP21A2 mutations were identified in both alleles, in two 21OHD patients CYP21A2 mutations were identified only in one allele. In four patients, mutation was not detected at all. In total, seventeen known and one novel, disease causing CYP21A2 mutations were observed. Among identified mutations, previously described c.293-13C/A>G, large rearrangements and p.Q319X mutations were the most common mutations accounting for 33.3%, 14.4% and 12.2% of all evaluated chromosomes, respectively. In six families (13.3%) a novel founder mutation, c.2T>C (p.M1?), inactivating the translation initiation codon was found. This mutation is not present in pseudogene CYP21A1P and causes the classical form of the disease in six patients. In addition, depending on the nature of the rearrangements CYP21A1P/CYP21A2 chimeras were further classified as CH^c/d, and CH-1^c was shown to be the most prominent chimera in our study group. In conclusion, with this study we identified a novel founder CYP21A2 mutation and suggest a further classification for CYP21A1P/CYP21A2 chimeras depending on the combination of junction site position and whether it is occurred as a result of deletion or conversion. Absence of disease causing mutation of CYP21A2 in ten of screened ninety chromosomes suggests the contribution of regulatory elements in occurrences of CAH due to the 21OHD.     

A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.