SMART 5: domains in the context of genomes and networks

The Simple Modular Architecture Research Tool (SMART) is an online resource () used for protein domain identification and the analysis of protein domain architectures. Many new features were implemented to make SMART more accessible to scientists from different fields. The new ‘Genomic’ mode in SMART makes it easy to analyze domain architectures in completely sequenced genomes. Domain annotation has been updated with a detailed taxonomic breakdown and a prediction of the catalytic activity for 50 SMART domains is now available, based on the presence of essential amino acids. Furthermore, intrinsically disordered protein regions can be identified and displayed. The network context is now displayed in the results page for more than 350 000 proteins, enabling easy analyses of domain interactions

Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

[EN] Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.IRO, VMZM, HHU and PLS were supported by the Mexican National Council of Science and Technology (CONACyT) with a PhD fellowship (210085, 210100, 243380 and 219883, respectively). Work in the SDF laboratory was financed by the CONACyT grants CB-2012-177739, FC-2015-2/1061, and INFR-2015-253504, and NMM by the CONACyT grant CB-2011-165986. SDF, CF and LC acknowledge the support of the European Union FP7-PEOPLE-2009-IRSES project EVOCODE (grant no. 247587) and H2020-MSCARISE-2015 project ExpoSEED (grant no. 691109). SDF also acknowledges the Marine Biological Laboratory (MBL) in Woods Hole for a scholarship for the Gene Regulatory Networks for Development Course 2015 (GERN2015). IE acknowledges the International European Fellowship-METMADS project and the Universita degli Studi di Milano (RTD-A; 2016). Research in the laboratory of MFY was funded by NSF (grant IOS-1121055), NIH (grant 1R01GM112976-01A1) and the Paul D. Saltman Endowed Chair in Science Education (MFY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Reyes Olalde, J.; Zuñiga, V.; Serwatowska, J.; Chávez Montes, R.; Lozano-Sotomayor, P.; Herrera-Ubaldo, H.; Gonzalez Aguilera, K.... (2017). The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium. PLoS Genetics. 13(4):1-31. https://doi.org/10.1371/journal.pgen.1006726S131134Reyes-Olalde, J. I., Zuñiga-Mayo, V. M., Chávez Montes, R. A., Marsch-Martínez, N., & de Folter, S. (2013). Inside the gynoecium: at the carpel margin. Trends in Plant Science, 18(11), 644-655. doi:10.1016/j.tplants.2013.08.002Alvarez-Buylla, E. 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ITAM-like signalling for efficient phagocytosis : The paradigm of the granulocyte receptor CEACAM3

Human CEACAM3 is a tailor-made receptor of the innate immune system to fight pathogens exploiting epithelial CEACAM-family members for colonisation and invasion of their host. Previous studies established CEACAM3 as the receptor facilitating rapid phagocytosis and elimination of N. gonorrhoeae by human granulocytes. The studies reported here set out to shed light on the evolution of this highly specialised receptor and the associated signalling machinery.CEACAM3 arose from exon shuffling after radiation of CEACAM1 approximately 24 million years ago in an ancestor of modern primates, bringing together the N-terminal domain of an epithelial CEACAM (CEACAM1, CEA or CEACAM6) targeted by pathogens with an intracellular signalling sequence most probably derived from an early CEACAM4 equipping it with a trigger of efficient phagocytosis. This signalling motif, that is closer to a canonical ITAM in CEACAM4, was further diversified during CEACAM3 evolution. Phagocytosis via CEACAM3 requires the activity of Src-family tyrosine kinases and activation of the small GTPase Rac, a regulator of the actin cytoskeleton. We could show, that phosphorylation of the tyrosines in the CEACAM3 ITAM-like sequence allows direct recruitment of the guanine nucleotide exchange-factor (GEF) Vav, which acts on Rac. Rac triggers lamellipodia formation by its effector, the WAVE-complex, a heteropentameric assembly providing interfaces for Rac and the adapter protein Nck via Sra1 and Nap1 respectively. We show, that CEACAM3 orchestrates activation as well as localisation of the actin-polymerisation machinery by recruitment of the WAVE-complex via Nck, which constitutively binds the WAVE-complex and is recruited to sites of CEACAM3 phosphorylation in a SH2-dependent manner. Further studies were undertaken to elucidate the role of additional kinases in the CEACAM3 signalling pathway. From similarities to immunoreceptor-signalling pathways the Tec and Syk family of tyrosine kinases were in the focus of those studies. While Tec kinases were found to be important in uptake and directly bind to pY230 of CEACAM3 during uptake, Syk associated with CEACAM3 wildtype only after uptake, but not close to the plasma membrane during the phagocytic process. This is in contrast to a CEACAM3/CD3ζ-ITAM chimera, where Syk co-localised with the chimera and bacteria in early uptake but not intracellular bacteria. Together, these studies provide novel insight into the protein-interaction network initiated by bacterial binding to CEACAM3 and help to explain the efficient and rapid phagocytosis mediated by this granulocyte receptor

CEACAMs : their role in physiology and pathophysiology

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulin-related glycoproteins. They are involved in cell cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated intemalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion

CEACAM3: An innate immune receptor directed against human-restricted bacterial pathogens

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related glycoprotein exclusively expressed on granulocytes. In contrast to other members of the CEACAM family, CEACAM3 does not support cell cell adhesion, but rather mediates the opsonin-independent recognition and elimination of a restricted set of human-specific Gram-negative bacterial pathogens including Neisseria gonorrhoeae, Haemophilus influenzae, and Moraxella catarrhalis. Within the last 4 years, molecular determinants of CEACAM3 function and CEACAM3-initiated signaling pathways have been elucidated. Sequence comparison between CEACAM3 and other CEACAM family members points to a chimeric origin of this receptor with the bacteriabinding extracellular domain and the function-promoting intracellular domain derived from different genes. This review summarizes the current knowledge about the structure function relationship of CEACAM3 and tries to combine these molecular aspects with a plausible scenario concerning the evolutionary origin of this phagocyte receptor in the light of host pathogen adaptation