Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino ...acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
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•Phase separation of FUS requires both the N-terminal PLD and the C-terminal RBD•Tyrosine and arginine govern the saturation concentration of phase separation•Glycine maintains liquidity, whereas glutamine and serine promote hardening•An associative polymer model predicts the phase behavior of FUS family proteins
The phase-separation behavior of FUS family proteins can be predicted by the prevalence and position of specific amino acids.
Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are ...unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors.
To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains.
FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Gluedare structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, ...we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2terminus of p150Gluedbinds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Gluedand LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.
Reconstitution of transmembrane proteins into supported lipid bilayer (SLB) membranes has often been hampered by strong interactions of protein domains with the underlying solid support leading to ...loss of both activity and mobility within the plane of the lipid bilayer. Polymer cushioned SLBs can overcome this by avoiding direct contact with the support. To extend this approach we developed an anionic polymer cushion system that allows tunable lipid mobility as well as functional integration of the transmembrane proteinβ-amyloid precursor protein cleaving enzyme (BACE) into SLBs. Fluorescence recovery after photobleaching analysis revealed a homogeneous distribution and high lateral mobility of the reconstituted BACE in cushioned SLBs while an impaired mobility and inhomogeneous clustering of reconstituted BACE were found in SLBs on silicon oxide substrates. The cushioning of SLBs led to increased incorporation and enhanced enzymatic activity of the reconstituted BACE with a direct correlation between lipid mobility and BACE activity. The utilized polymer cushion system allows the successful reconstitution of transmembrane proteins within SLBs with tunable properties.
Supported lipid bilayer membranes on polymer cushions improve unrestricted lateral mobility and enhance the functionality of integrated transmembrane proteins in comparison to solid supported bilayer systems.
Nutrients and growth hormones promote insulin production and the proliferation of pancreatic beta-cells. An imbalance between ever-increasing metabolic demands and insulin output causes diabetes. ...Recent evidence indicates that beta-cells enhance insulin gene expression depending on their secretory activity. This signalling pathway involves a catalytically inactive receptor tyrosine phosphatase, ICA512, whose cytoplasmic tail is cleaved on glucose-stimulated exocytosis of insulin secretory granules and then moves into the nucleus, where it upregulates insulin transcription. Here, we show that the cleaved cytosolic fragment of ICA512 enhances the transcription of secretory granule genes (including its own gene) by binding to tyrosine phosphorylated signal transducers and activators of transcription (STAT) 5 and preventing its dephosphorylation. Sumoylation of ICA512 by the E3 SUMO ligase PIASy, in turn, may reverse this process by decreasing the binding of ICA512 to STAT5. These findings illustrate how the exocytosis of secretory granules, through a retrograde pathway that sustains STAT activity, converges with growth hormone signalling to induce adaptive changes in beta-cells in response to metabolic demands.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
This paper reports on performances of high permeability grain oriented electrical steel when used in association with power electronic switching devices. Loss measurement results obtained from the ...Epstein test, using sinusoidal or various PWM voltages in medium frequency range, show that for both studied thicknesses (HGO 0.23mm and HGO 0.18mm), comparing performances at a fixed induction level between the various situations may not be the most convenient method. The effect of magnetic domain refinement has been investigated. After having shown the interest of lowering the thickness, an alternative way of looking at losses is proposed that may help to design the magnetic core when it comes to the matter of reducing size in considering frequency and magnetization levels.
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•Design of a SST cell to ensure a good technical-economic balance has been presented.•Paving the way for the construction of SST made of Grain-Oriented Electrical steel.•Ensure a ...galvanic isolation compatible with medium voltage electrical networks.•Provide a good thermal balance of the MFT compatible with Class H (180°C–2000 h).•Development of the control and regulation system of two SST cells.
The increasing part of intermittent renewable energy requires significant modifications of the electric grid. The introduction of smart nodes, where the power transfer can be adjusted in real time in addition to the classical voltage change and the galvanic isolation, becomes an important asset for the grid balance. Transformers associated with power electronic converters can ensure this function; they are known generically as Solid-State Transformers (SST). Their high specific power also opens opportunities for railway application. SSTs can be operated at high frequencies (a few tens of kHz), with ferrite or nanocrystalline cores operating at low flux densities. The paper proposes a complementary approach that consists in using transformers built with grain oriented electrical steel (GOES) wound cores, operating at medium frequencies (few kHz) and high flux densities. The reported work focuses on experimental assessment aiming to show that this option is now open for designing SST cells, which can be associated to reach bigger units.
Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it ...specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.
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•The organelle content of the Balbiani body is held together by an Xvelo matrix•Xvelo forms amyloid-like networks in vitro, which can recruit RNA and mitochondria•Prion-like domain of Xvelo dictates specificity in amyloid assembly•Amyloid-like polymerization is conserved among vertebrate Balbiani body organizers
Amyloid-like self-assembly of a specific protein drives formation of a cellular compartment in oocytes.
SignificanceBiomolecular condensates are intracellular organelles that are not bounded by membranes and often show liquid-like, dynamic material properties. They typically contain various types of ...proteins and nucleic acids. How the interaction of proteins and nucleic acids finally results in dynamic condensates is not fully understood. Here we use optical tweezers and fluorescence microscopy to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) condenses with individual molecules of single- and double-stranded DNA. We find that FUS adsorbs on DNA in a monolayer and hence generates an effectively sticky FUS-DNA polymer that collapses and finally forms a dynamic, reversible FUS-DNA co-condensate. We speculate that protein monolayer-based protein-nucleic acid co-condensation is a general mechanism for forming intracellular membraneless organelles.
Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand ...how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.
•Pseudotyped baculovirus (BV) efficiently transfects axolotl intact limb tissue.•High efficient transfection is achieved in limb blastema.•PRRX1+ and PAX7+ cells are efficiently transfected and participate in regeneration.•BV elicits early and strong transgene expression.•BV is a complementary tool to current gene transfer methods.
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