Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the ...DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
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•Caspase-8 tDED assembles into filaments through quasi-equivalent contacts•The assembly of caspase-8 filaments is nucleated by the upstream Fas/FADD complex•cFLIP tDED also forms filaments, which interact with caspase-8 by comingling•MC159 inhibits caspase-8 filament assembly by a unique capping mechanism
How caspase-8 is activated has been a long-standing question. Fu et al. show that its tDED forms filaments using quasi-equivalent interactions. Cryo-EM structure of the filament reveals mechanisms of caspase-8 activation and its regulation by cFLIP and MC159.
CarD, an essential transcription regulator in Mycobacterium tuberculosis , directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish ...that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn–/– mice exhibit a global ...deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn–/– brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN—a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn–/– phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn–/– CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
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•Grn–/– mice exhibit robust BMP lipid deficiency with concurrent GlcSph accumulation•BMP stimulates glucocerebrosidase activity and lysosome function in Grn–/– models•PGRN was fused to a human transferrin receptor binding Fc to increase CNS exposure•PTV:PGRN biologic rescues Grn–/– CNS lysosomal, microglial, and neuronal dysfunction
Peripherally delivered progranulin biologic with enhanced CNS biodistribution corrects CNS disease pathology of the GRN mouse model of frontotemporal dementia.
H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to ...targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.
Abstract
Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport ...vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TV
CD98hc
). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATV
CD98hc
) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATV
CD98hc
demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATV
CD98hc
that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TV
CD98hc
as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.
Transcription-repair coupling factor (TRCF; the product of the
mfd gene) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNA polymerase stalled at ...a noncoding lesion in the DNA template strand, uses the energy from ATP hydrolysis to disrupt the transcription complex, and stimulates DNA repair by recruiting UvrA, a component of the nucleotide excision repair machinery, to the site. TRCF is a large (130 kDa) multifunctional protein with a complex structure–function relationship consisting of a compact arrangement of eight structured domains linked by flexible linkers. Through a conserved, intramolecular, interdomain interaction, TRCF is held in a conformation in which its enzymatic activities (ATPase activity and DNA translocase activity) are strongly repressed. Disruption of the repressive interdomain interaction by amino acid substitutions within the interface derepresses ATPase and DNA translocase activities. In this work, we have shown that derepressed TRCF mutants are dramatically sensitized to limited proteolysis compared with repressed TRCF, pointing to an altered conformational state. Analysis of the protease cleavage sites mapped onto the structure of the repressed TRCF conformation indicates that (1) the cleavage sites tend to cluster at linkers connecting the TRCF structured domains, and (2) many of the cleavage sites sensitized in the derepressed TRCF are partially or completely buried to protease access in the repressed TRCF structure. We conclude that TRCF derepression is associated with profound conformational changes that primarily involve a reorganization of the interdomain interactions.
A 40 kDa glycoprotein (SPG‐40) secreted during involution works as a protective signalling factor through its binding to viable cells. The crystal structure of the native protein has been determined ...at 2.3 Å resolution. This is the first report on the carbohydrate‐binding properties of SPG‐40; the structure determinations of the complexes of SPG‐40 with four oligosaccharides of different lengths at resolutions ranging from 2.2 to 2.8 Å are described. Carbohydrate‐binding studies with N‐acetylglucosamines (GlcNAcn, n = 3–6) using fluorescence spectroscopy revealed poor binding effects with GlcNAc3 and GlcNAc4, while GlcNAc5 and GlcNAc6 bound to SPG‐40 with considerable strength; the dissociation constants (Kd) were estimated to be 260 ± 3 and 18 ± 4 µM, respectively. SPG‐40 was cocrystallized with GlcNAc3, GlcNAc4, GlcNAc5 and GlcNAc6. The overall structure of native SPG‐40 was essentially similar to that reported previously at low resolution. The structures of its complexes with GlcNAc3, GlcNAc4, GlcNAc5 and GlcNAc6 revealed the positions of these oligosaccharides in the carbohydrate‐binding groove and provided insights into the mechanism of binding of oligosaccharides to SPG‐40, indicating that the preferred subsites in the carbohydrate‐binding groove of SPG‐40 were from −4 to −2. The structure of the protein remained unperturbed upon binding of GlcNAc3 and GlcNAc4, but the structure changed significantly upon binding of GlcNAc5 and GlcNAc6. Significant conformational variations were observed in the sugar‐binding groove: Trp78 partially flipped out of the barrel in GlcNAc5, while in the GlcNAc6 complex a completely flipped‐out Trp78 was observed along with several other conformational changes, including those of Asp186 and Arg242. Such changes upon binding to carbohydrates have not previously been observed in chitin‐hydrolyzing chitinases and reflect less favourable binding of carbohydrates to SPG‐40. As this appears to essentially be a binding protein, this loss of binding affinity might be compensated by other intermolecular interactions such as protein–protein interactions and also by the binding of its own glycan chain.
A recently discovered new class of 40 kDa glycoproteins forms a major component of the secretory proteins in the dry secretions of non-lactating animals. These proteins are implicated as protective ...signalling factors that determine which cells are to survive during the processes of drastic tissue remodelling. In order to understand its role in the remodelling of mammary glands, the detailed three-dimensional structure of the bovine signalling glycoprotein (SPC-40) has been determined using X-ray crystallography. SPC-40 was purified from bovine dry secretions and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.6, b = 67.4, c = 106.9 Angstrom. The protein was also cloned in order to determine its complete amino-acid sequence. Its three-dimensional structure has been determined using data to 2.1 Angstrom resolution. The amino-acid sequence determination of SPC-40 reveals two potential N-glycosylation sites at Asn39 and Asn345, but electron density for a glycan chain was only present at Asn39. The protein adopts a conformation with the classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM barrel; residues 1-237 and 310-360) with the insertion of a small alpha+beta domain (residues 240-307) similar to that observed in chitinases. However, the substitution of Leu for Glu in the consensus catalytic sequence in SPC-40 caused a loss of chitinase activity. Furthermore, the chitin-binding groove in SPC-40 is considerably distorted owing to unfavourable conformations of several residues, including Trp78, Tyr120, Asp186 and Arg242. Three surface loops, His188-His197, Phe202-Arg212 and Tyr244-Pro260, have exceptionally high B factors, suggesting large-scale flexibility. Fluorescence studies indicate that various sugars bind to SPC-40 with low affinities.
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