Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products.     

Structural basis for RanGTP independent entry of spliceosomal U snRNPs into the nucleus

The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importinβ (Impβ) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Impβ/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Impβ (127-876) in complex with the Impβ-binding (IBB) domain of SPN1 (1-65) at 2.8-Å resolution reveals that Impβ adopts an open conformation, which is unique for a functional Impβ/cargo complex, and rather surprisingly, it resembles the conformation of the Impβ/RanGTP complex. As binding of RanGTP to Impβ usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Impβ/RanGTP the independent dissociation of Impβ/SPN1 from the nuclear basket is energetically aided.     

Molecular characterization of the Rpt1/p48B ATPase subunit of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> 26S proteasome

The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5′-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic effect of the mutations affecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.     

Structural Basis for Recognition of Ubiquitylated Nucleosome by Dot1L Methyltransferase

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Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.     

Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD⁺-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD⁺-bound LDH are reported at 3.0 and 3.2?Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD⁺. NAD⁺ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD⁺-bound form was approximately three times higher than that of the apo form after incubation at 80?°C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.     

MiR‐377 inhibits wear particle‐induced osteolysis via targeting RANKL

Periprosthetic osteolysis caused by wear particles is the main factor that affects the long‐term efficacy of artificial joint replacement, and macrophages play a vital role in the pathogenesis of periprosthetic osteolysis, while the potential mechanism underlying this is still unclear. To investigate the underlying role of miR‐377 in wear particle‐induced osteolysis (PIO), blood samples from patients undergoing arthroplasty were collected for analyzing the correlation between miR‐377 expression and the clinicopathological parameters of PIO. Peripheral blood macrophages were obtained to compare the miR‐377 and receptor activator of NF‐κB ligand (RANKL) expressions. Bone marrow macrophages (BMMs) following titanium (Ti) particle treatment and/or miR‐377 mimic transfection were used. The expressions of RANKL, pro‐inflammatory cytokines interleukin 6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) and the osteoclast‐related molecules tartrate‐resistant acid phosphatase (TRAP) and cathepsin K (CTSK) were determined using real‐time polymerase chain reaction or western blotting or enzyme‐linked immunosorbent assay or TRAP staining, when appropriate. The interaction between miR‐377 and RANKL was assessed by luciferase reporter assay. The in vivo role of miR‐377 in PIO was evaluated using a mouse calvarial osteolysis model. There were significant differences in downregulated miR‐377 expression between the different numbers of particles in the joint prostheses. The Ti particle treatment increased pro‐inflammatory cytokine levels, downregulated RANKL and increased osteoclast activity in BMMs, while miR‐377 overexpression led to the opposite effect. Taken together, miR‐377 downregulated the target gene RANKL, resulting in PIO inhibition. MiR‐377 relieved PIO by negatively regulating RANKL.     

Experimental Study of Aqueous Humor Flow in a Transparent Anterior Segment Phantom by Using PIV Technique

Pupillary block is considered as an important cause of primary angle-closure glaucoma (PACG). In order to investigate the effect of pupillary block on the hydrodynamics of aqueous humor (AH) in anterior chamber (AC) and potential risks, a 3D printed eye model was developed to mimic the AH flow driven by fluid generation, the differential pressure between AC and posterior chambers (PC) and pupillary block. Particle image velocimetry technology was applied to visualize flow distribution. The results demonstrated obvious differences in AH flow with and without pupillary block. Under the normal condition (without pupillary block), the flow filed of AH was nearly symmetric in the AC. The highest flow velocity located at the central of AC when the differential pressure between AC and PC was under 5.83 Pa, while it appeared near the cornea and iris surface when the differential pressure was greater than 33.6 Pa. Once pupillary block occurred, two asymmetric vortices with different sizes were observed and the shear stress in the paracentral cornea and iris epithelium increased greatly. It can be concluded that the pupillary block and the elevated differential pressure between AC and PC could change the flow distribution and thus increase the risk of corneal endothelial cells detachment. This study could make a further understanding of the pathogenesis of PACG.