Ptk7-Deficient Mice Have Decreased Hematopoietic Stem Cell Pools as a Result of Deregulated Proliferation and Migration

International audienceHematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. Atthe top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features arecontrolled through key microenvironmental cues and regulatory pathways, such as Wnt signaling.We showed previously that PTK7,a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesishas remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, withthe highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss ofPtk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlatedwith increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling noveland unexpected functions for planar cell polarity pathways in HSC fate

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance

Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.journal article2014 Mar 182014 03 18importe

The Human PDZome 2.0: Characterization of a New Resource to Test for PDZ Interactions by Yeast Two-Hybrid

PSD95-disc large-zonula occludens (PDZ) domains are globular modules of 80–90 amino acids that co-evolved with multicellularity. They commonly bind to carboxy-terminal sequences of a plethora of membrane-associated proteins and influence their trafficking and signaling. We previously built a PDZ resource (PDZome) allowing us to unveil human PDZ interactions by Yeast two-hybrid. Yet, this resource is incomplete according to the current knowledge on the human PDZ proteome. Here we built the PDZome 2.0 library for Yeast two-hybrid, based on a PDZ library manually curated from online resources. The PDZome2.0 contains 305 individual clones (266 PDZ domains in isolation and 39 tandems), for which all boundaries were designed based on available PDZ structures. Using as bait the E6 oncoprotein from HPV16, a known promiscuous PDZ interactor, we show that PDZome 2.0 outperforms the previous resource

PDZ scaffolds regulate extracellular vesicle production, composition, and uptake

International audienceExtracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome ( ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication

Syntenin mediates SRC function in exosomal cell-to-cell communication

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C2orf62 and TTC17 are involved in actin organization and ciliogenesis in zebrafish and human.

Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis

Syntenin-knock out reduces exosome turnover and viral transduction

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Tetraspanin-6 negatively regulates exosome production

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The unique Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. The mechanisms controlling nuclear protein localisation in the context of stress responses induced upon bacterial infection are still poorly understood. Here we show that the Brucella abortus effectors NyxA and NyxB interfere with the host sentrin specific protease 3 (SENP3), which is essential for intracellular replication. Translocated Nyx effectors directly interact with SENP3 via a defined acidic patch identified from the crystal structure of NyxB, preventing its nucleolar localisation at the late stages of the infection. By sequestering SENP3, the Nyx effectors induce the cytoplasmic accumulation of the nucleolar AAA-ATPase NVL, the large subunit ribosomal protein L5 (RPL5) and the ribophagy receptor NUFIP1 in Nyx-enriched structures in the vicinity of replicating bacteria. This shuttling of ribosomal biogenesis-associated nucleolar proteins is negatively regulated by SENP3 and dependent on the autophagy-initiation protein Beclin1, indicative of a ribophagy-derived process induced during Brucella infection. Our results highlight a new nucleomodulatory function by two unique Brucella effectors, and reveal that SENP3 is a critical regulator of the subcellular localisation of multiple nucleolar proteins during Brucella infection, promoting intracellular replication