Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP.

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

Immunization of Mice with Virus-Like Vesicles of Kaposi Sarcoma-Associated Herpesvirus Reveals a Role for Antibodies Targeting ORF4 in Activating Complement-Mediated Neutralization.

Infection by Kaposi sarcoma-associated herpesvirus (KSHV) can cause severe consequences, such as cancers and lymphoproliferative diseases. Whole inactivated viruses (WIV) with chemically destroyed genetic materials have been used as antigens in several licensed vaccines. During KSHV productive replication, virus-like vesicles (VLVs) that lack capsids and viral genomes are generated along with virions. Here, we investigated the immunogenicity of KSHV VLVs produced from a viral mutant that was defective in capsid formation and DNA packaging. Mice immunized with adjuvanted VLVs generated KSHV-specific T cell and antibody responses. Neutralization of KSHV infection by the VLV immune serum was low but was markedly enhanced in the presence of the complement system. Complement-enhanced neutralization and complement deposition on KSHV-infected cells was dependent on antibodies targeting viral open reading frame 4 (ORF4). However, limited complement-mediated enhancement was detected in the sera of a small cohort of KSHV-infected humans which contained few neutralizing antibodies. Therefore, vaccination that induces antibody effector functions can potentially improve infection-induced humoral immunity. Overall, our study highlights a potential benefit of engaging complement-mediated antibody functions in future KSHV vaccine development. IMPORTANCE KSHV is a virus that can lead to cancer after infection. A vaccine that prevents KSHV infection or transmission would be helpful in preventing the development of these cancers. We investigated KSHV VLV as an immunogen for vaccination. We determined that antibodies targeting the viral protein ORF4 induced by VLV immunization could engage the complement system and neutralize viral infection. However, ORF4-specific antibodies were seldom detected in the sera of KSHV-infected humans. Moreover, these human sera did not potently trigger complement-mediated neutralization, indicating an improvement that immunization can confer. Our study suggests a new antibody-mediated mechanism to control KSHV infection and underscores the benefit of activating the complement system in a future KSHV vaccine.

Static and dynamic disorder in Aß40 fibrils.

Deposition of Aß aggregates in the form of amyloid fibrils is a pathological hallmark of Alzheimer's disease. Understanding the structure and dynamics of Aß fibrils is important for delineating the mechanism of Aß aggregation and developing effective therapeutic strategies. Here we used site-directed spin labeling and EPR spectroscopy to study the Aß40 fibril structure and dynamics. We obtained the EPR spectra of 40 spin-labeled Aß40 fibril samples, with spin labeling coverage of the entire Aß40 sequence. Analysis of the spin exchange interaction and spin label mobility using spectral simulations suggest that the strength of spin exchange interaction is primarily determined by static disorder in the Aß40 fibrils. EPR data suggest that the entire Aß40 sequence except residue D1 is highly ordered and the two hydrophobic regions at residues 17-20 and 31-36 show the lowest static disorder. Dynamic disorder is relatively constant across all reside positions, with residues 22 and 23 having the highest dynamic disorder. Comparison of the EPR data for Aß40 and Aß42 fibrils shows overall more ordered packing interactions in Aß40 fibrils. Another noteworthy difference is the C-terminal residue, which has high static disorder in Aß42 fibrils, but is ordered in Aß40 fibrils. The higher static disorder in Aß42 fibrils may lead to increased fragmentation, monomer dissociation, and structural defects, which may contribute to increased aggregation through secondary nucleation.

Segmental structural dynamics in Aß42 globulomers.

Aß42 aggregation plays a central role in the pathogenesis of Alzheimer's disease. In addition to the insoluble fibrils that comprise the amyloid plaques, Aß42 also forms soluble aggregates collectively called oligomers, which are more toxic and pathogenic than fibrils. Understanding the structure and dynamics of Aß42 oligomers is critical for developing effective therapeutic interventions against these oligomers. Here we studied the structural dynamics of Aß42 globulomers, a type of Aß42 oligomers prepared in the presence of sodium dodecyl sulfate, using site-directed spin labeling. Spin labels were introduced, one at a time, at all 42 residue positions of Aß42 sequence. Electron paramagnetic resonance spectra of spin-labeled samples reveal four structural segments based on site-dependent spin label mobility pattern. Segment-1 consists of residues 1-6, which have the highest mobility that is consistent with complete disorder. Segment-3 is the most immobilized region, including residues 31-34. Segment-2 and -4 have intermediate mobility and are composed of residues 7-30 and 35-42, respectively. Considering the inverse relationship between protein dynamics and stability, our results suggest that residues 31-34 are the most stable segment in Aß42 oligomers. At the same time, the EPR spectral lineshape suggests that Aß42 globulomers lack a well-packed structural core akin to that of globular proteins.

Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney.

The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in native states remain unknown. Here, we report the cryo-electron microscopy structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at resolutions of 3.5 Å and 3.8 Å, respectively. The structures of multiple proteins were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, the native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. In addition, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the twofold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides a direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.

Screening Tests for Lasso Problems.

This paper is a survey of dictionary screening for the lasso problem. The lasso problem seeks a sparse linear combination of the columns of a dictionary to best match a given target vector. This sparse representation has proven useful in a variety of subsequent processing and decision tasks. For a given target vector, dictionary screening quickly identifies a subset of dictionary columns that will receive zero weight in a solution of the corresponding lasso problem. These columns can be removed from the dictionary prior to solving the lasso problem without impacting the optimality of the solution obtained. This has two potential advantages: it reduces the size of the dictionary, allowing the lasso problem to be solved with less resources, and it may speed up obtaining a solution. Using a geometrically intuitive framework, we provide basic insights for understanding useful lasso screening tests and their limitations. We also provide illustrative numerical studies on several datasets.

Edge-preserving image regularization based on morphological wavelets and dyadic trees.

Despite the tremendous success of wavelet-based image regularization, we still lack a comprehensive understanding of the exact factor that controls edge preservation and a principled method to determine the wavelet decomposition structure for dimensions greater than 1. We address these issues from a machine learning perspective by using tree classifiers to underpin a new image regularizer that measures the complexity of an image based on the complexity of the dyadic-tree representations of its sublevel sets. By penalizing unbalanced dyadic trees less, the regularizer preserves sharp edges. The main contribution of this paper is the connection of concepts from structured dyadic-tree complexity measures, wavelet shrinkage, morphological wavelets, and smoothness regularization in Besov space into a single coherent image regularization framework. Using the new regularizer, we also provide a theoretical basis for the data-driven selection of an optimal dyadic wavelet decomposition structure. As a specific application example, we give a practical regularized image denoising algorithm that uses this regularizer and the optimal dyadic wavelet decomposition structure.