The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3′ to 5′ Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit ...Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
Display omitted
•Structure of a human nuclear exosome-MTR4-RNA complex at 3.45 Å overall resolution•DNA-RNA chimera translocation strand can stall the MTR4 helicase•Mtr4 helicase activities are stimulated in yeast and human exosomes•Mutual exclusivity among exosome cofactors implies a sequential process for RNA decay
The cryo-EM structure of the human nuclear exosome-Mtr4 complex loaded with an RNA substrate reveals mechanistic insights to how the helicase unwinds structured RNA substrates to promote degradation.
Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. ...Furthermore, α-conotoxins (α-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3β2 and α3β4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R
of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3β2 and α3β4 nAChR homology models bound to the α-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 ± 0.08 kcal/mol and an R
of 0.49. This is significantly better than the widely used molecular mechanics-generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 ± 0.24 kcal/mol and an R
of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3β2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the α3β2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.
Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The ...discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1-blocking agents. Herein we report a de novo missense mutation (c.1009A > T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4-transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Activated STING in a Vascular and Pulmonary Syndrome Liu, Yin; Jesus, Adriana A; Marrero, Bernadette ...
New England journal of medicine/The New England journal of medicine,
08/2014, Letnik:
371, Številka:
6
Journal Article
Recenzirano
Odprti dostop
The stimulator of interferon genes (STING) protein bridges sensors of cytosolic DNA and the inflammatory pathway mediated by interferon-β. Activating mutations in the
STING
gene cause a vascular and ...pulmonary syndrome.
Studies involving children with monogenic autoinflammatory disease have provided insights into the regulation of key proinflammatory cytokine pathways and their role in causing systemic and organ-specific inflammation.
1
,
2
We studied six patients who presented in early infancy with systemic inflammation and violaceous, scaling lesions of fingers, toes, nose, cheeks, and ears that progressed to acral necrosis in most of the patients and did not respond to therapy. A mixed histologic pattern was observed, consisting of a prominent dermal inflammatory infiltrate with features of leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels. Three of the patients had severe interstitial lung . . .
As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid ...(SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the βHI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus.
Despite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.
Chronic hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a nonparticulate variant of ...the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. Here, we report the crystal structure of HBeAg, which clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerization relative to HBcAg (∼140° rotation), locked into place through formation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T cell level (through sequence identity) but not at the B cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.
▸ We report the structure of HBV e-antigen, a non-particulate variant of capsid protein ▸ HBeAg's mode of dimerization is radically different than that of capsid protein ▸ Alternative dimerization is mediated by an HBeAg-specific intramolecular disulfide ▸ HBeAg is implicated in immune tolerance and chronic hepatitis B infection
Hepatitis B virus expresses two forms of capsid protein, a core antigen, HBcAg, which forms the capsid, and an e-antigen, HBeAg, which is implicated in establishing immune tolerance and chronic infection. DiMattia et al. determine HBeAg structure and explain the biophysical and antigenic differences between them.
HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the ...host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the “A”-shaped architecture of the RRE in export-active complexes.
Display omitted
•Rev NTD α-helical hairpins dimerize with widely varying crossing angles•The unfolded CTDs become partially ordered when Rev assembles into helical filaments•Cryo-EM of filaments and crystal structures reveal a novel inter-molecular interface•This interface and variations in crossing angle may help organize Rev-RRE complexes
Binding of 8–12 HIV-1 Rev subunits to the RRE of viral transcripts mediates nuclear export. DiMattia et al. describe a novel Rev-Rev interface and the remarkably flexible ways in which Rev can assemble, and propose how this allows Rev to engage with the A-shaped RRE.
PDF
