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The endoplasmic reticulum (ER) is a major site for membrane protein synthesis in eukaryotes. The majority of integral membrane proteins are delivered to the ER membrane via the co-translational, signal recognition particle (SRP)-dependent route. However, tail-anchored proteins employ an alternative, post-translational route(s) that relies on distinct factors such as a cytosolic protein quality control component, SGTA. We now show that SGTA is selectively recruited to ribosomes synthesising a diverse range of membrane proteins, suggesting that its biosynthetic client base also includes precursors on the co-translational ER delivery pathway. Strikingly, SGTA is recruited to nascent membrane proteins before their transmembrane domain emerges from the ribosome. Hence, SGTA is ideally placed to capture these aggregation prone regions shortly after their synthesis. For nascent membrane proteins on the co-translational pathway, SGTA complements the role of SRP by reducing the co-translational ubiquitination of clients with multiple hydrophobic signal sequences. On this basis, we propose that SGTA acts to mask specific transmembrane domains located in complex membrane proteins until they can engage the ER translocon and become membrane inserted.
Hundreds of nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae, the cytosol has the capacity to transiently store mitochondrial matrix-destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus-encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins.
RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site - contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.
One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer.
DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
During development, neural precursors divide to produce new precursors and cells that differentiate as neurons and glia. In Drosophila, apicobasal polarity and orientation of the mitotic spindle play important roles in specifying the progeny of neural precursors for different fates. We examined orientation of zebrafish spinal cord precursors using time-lapse imaging and tested the function of protein kinase C, iota (PrkCi), a member of the Par complex of proteins necessary for apicobasal polarity in the nervous system. We found that nearly all precursors divide within the plane of the neuroepithelium of wild-type embryos even when they must produce cells that have different fates. In the absence of PrkCi function, neural precursor divisions become oblique during late embryogenesis and excess oligodendrocytes form concomitant with loss of dividing cells. We conclude that PrkCi function and planar divisions are necessary for asymmetric, self-renewing division of spinal cord precursors.
Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates. The HLH domain mediates formation of homo- or heterodimers. We have taken advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system. Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole. During germ band retraction these cells spread along the future midgut region. Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature. Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles.
Current models propose that group 2 innate lymphoid cells (ILC2s) are generated in the bone marrow. Here, we demonstrate that subsets of these cells can differentiate from multipotent progenitors and committed T cell precursors in the thymus, both in vivo and in vitro. These thymic ILC2s exit the thymus, circulate in the blood, and home to peripheral tissues. Ablation of E protein transcription factors greatly promotes the ILC fate while impairing B and T cell development. Consistently, a transcriptional network centered on the ZBTB16 transcription factor and IL-4 signaling pathway is highly up-regulated due to E protein deficiency. Our results show that ILC2 can still arise from what are normally considered to be committed T cell precursors, and that this alternative cell fate is restrained by high levels of E protein activity in these cells. Thymus-derived lung ILC2s of E protein-deficient mice show different transcriptomes, proliferative properties, and cytokine responses from wild-type counterparts, suggesting potentially distinct functions.
Vasculogenesis describes the process of de novo vessel formation from vascular precursor cells. Although formation of the first major vessels, such as the dorsal aorta and cardinal veins, occurs during embryonic vasculogenesis, the contribution of precursor cell populations to postnatal vessel development is not well understood. Here, we identified a novel population of postnatal vascular precursor cells in mice. These cells express the Schwann cell protein myelin protein zero (Po) and exhibit a CD45(-)CD31(-)VEcad(-)c-kit(+)CXCR4(+) surface phenotype. Po(+) vascular precursors (PVPs) are recruited into the growing vasculature, and comprise a minor population of arterial endothelial cells in adult mice. Recruitment of PVPs into growing vessels is mediated by CXCL12-CXCR4 signaling, and is enhanced during vascular expansion induced by Notch inhibition. Po-specific ablation of Flk1, a receptor for VEGF, results in branching defects and insufficient arterial patterning in the retina, as well as reduced neovascularization of tumors and ischemic tissues. Thus, in postnatal mice, although growing vessels are formed primarily by angiogenesis from preexisting vessels, a minor population of arterial endothelia may be derived from tissue-resident vascular precursor cells.
Protein Tyrosine Phosphatase gamma (PTPgamma) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPgamma is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPgamma mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPgamma specific antibody that recognizes the protein by flow cytometry. PTPgamma expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPgamma was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34(+) haematopoietic precursors express high PTPgamma level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPgamma as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.
The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach.
A single asymmetric division by an adult neural stem cell (NSC) ultimately generates dozens of differentiated progeny, a feat made possible by the proliferative expansion of transit-amplifying progenitor cells (TAPs). Although NSC activation and TAP expansion is determined by pro- and anti-proliferative signals found within the niche, remarkably little is known about how these cells integrate simultaneous conflicting signals. We investigated this question focusing on the subventricular zone (SVZ) niche of the adult murine forebrain. Using primary cultures of SVZ cells, we demonstrate that Epidermal Growth Factor (EGF) and Bone Morphogenetic Protein (BMP)-2 are particularly powerful pro- and anti-proliferative factors for SVZ-derived neural precursors. Dose-response experiments showed that when simultaneously exposed to both signals, BMP dominantly suppressed EGF-induced proliferation; moreover, this dominance extended to all parameters of neural precursor behavior tested, including inhibition of proliferation, modulation of cell cycle, promotion of differentiation, and increase of cell death. BMP's anti-proliferative effect did not involve inhibition of mTORC1 or ERK signaling, key mediators of EGF-induced proliferation, and had distinct stage-specific consequences, promoting TAP differentiation but NSC quiescence. In line with these in vitro data, in vivo experiments showed that exogenous BMP limits EGF-induced proliferation of TAPs while inhibition of BMP-SMAD signaling promotes activation of quiescent NSCs. These findings clarify the stage-specific effects of BMPs on SVZ neural precursors, and support a hierarchical model in which the anti-proliferative effects of BMP dominate over EGF proliferation signaling to constitutively drive TAP differentiation and NSC quiescence.
NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors.
Langerhans cells are characterized by specific markers, such as Birbeck granules and CD1a antigen. S100 protein and neuron-specific enolase are less specific but are expressed only on Langerhans cells among the cells of the phagocytic mononuclear system. In this study, the expression of these two antigens on many monocytic leukemic cells is shown. These cells could be precursors of Langerhans cells which have been transformed into malignant cells.
Neuropeptides play critical roles in many biological processes such as growth, learning, memory, metabolism, and neuronal differentiation. A few approaches have been reported for predicting neuropeptides that are cleaved from precursor protein sequences. However, these models for cleavage site prediction of precursors were developed using a limited number of neuropeptide precursor datasets and simple precursors representation models. In addition, a universal method for predicting neuropeptide cleavage sites that can be applied to all species is still lacking. In this paper, we proposed a novel deep learning method called DeepNeuropePred, using a combination of pre-trained language model and Convolutional Neural Networks for feature extraction and predicting the neuropeptide cleavage sites from precursors. To demonstrate the model's effectiveness and robustness, we evaluated the performance of DeepNeuropePred and four models from the NeuroPred server in the independent dataset and our model achieved the highest AUC score (0.916), which are 6.9%, 7.8%, 8.8%, and 10.9% higher than Mammalian (0.857), insects (0.850), Mollusc (0.842) and Motif (0.826), respectively. For the convenience of researchers, we provide a web server (http://isyslab.info/NeuroPepV2/deepNeuropePred.jsp).
We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import.
In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.
Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome.
Dried porous materials based on plant proteins are attracting large attention thanks to their potential use as sustainable food ingredients. Nevertheless, plant proteins present lower gelling properties than animal ones. Plant protein gelling could be improved by optimising gelation conditions by acting on protein concentration, pH, and ionic strength. This work aimed to systematically study the effect of these factors on the gelation behaviour of soy and pea protein isolates. Protein suspensions having different concentrations (10, 15, and 20% w/w), pH (3.0, 4.5, 7.0), and ionic strength (IS, 0.0, 0.6, 1.5 M) were heat-treated (95 °C for 15 min) and characterised for rheological properties and physical stability. Strong hydrogels having an elastic modulus (G') higher than 103 Pa and able to retain more than 90% water were only obtained from suspensions containing at least 15% soy protein, far from the isoelectric point and at an IS above 0.6 M. By contrast, pea protein gelation was achieved only at a high concentration (20%), and always resulted in weak gels, which showed increasing G' with the increase in pH and IS. Results were rationalised into a map identifying the gelation conditions to modulate the rheological properties of soy and pea protein hydrogels, for their subsequent conversion into xerogels, cryogels, and aerogels.
Children with Tourette syndrome (TS) often have comorbid disorders, particularly attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD). While subtle premorbid symptoms have been described in various psychiatric disorders, the presence of clinical precursors that may exist before the onset of tics is unknown. This longitudinal study aimed to find clinical precursors of tics by assessing a range of clinical characteristics prior to tic onset in comparison with children without onset of tics.
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