Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

SummaryThe ring-shaped cohesin complex brings together distant DNA domains to maintain, express, and segregate the genome. Establishing specific chromosomal linkages depends on cohesin recruitment to defined loci. One such locus is the budding yeast centromere, which is a paradigm for targeted cohesin loading. The kinetochore, a multiprotein complex that connects centromeres to microtubules, drives the recruitment of high levels of cohesin to link sister chromatids together. We have exploited this system to determine the mechanism of specific cohesin recruitment. We show that phosphorylation of the Ctf19 kinetochore protein by a conserved kinase, DDK, provides a binding site for the Scc2/4 cohesin loading complex, thereby directing cohesin loading to centromeres. A similar mechanism targets cohesin to chromosomes in vertebrates. These findings represent a complete molecular description of targeted cohesin loading, a phenomenon with wide-ranging importance in chromosome segregation and, in multicellular organisms, transcription regulation.

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.DOI: http://dx.doi.org/10.7554/eLife.06057.001

Accurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins-Mif2/CENP-C and Chl4/CNEP-N-interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae, form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation.

The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. M pro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 M pro bound to two viral substrate peptides. The structures show how M pro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 M pro cleavage sites but conserved among homologous junctions in coronaviruses. M pro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of M pro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

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Dengue virus (DENV) is a mosquito-borne pathogen that is the causative agent of dengue fever. Severe dengue virus infection is potentially fatal due to hemorrhaging, plasma leakage, and pulmonary shock. The four serotypes of DENV (DENV-1 to DENV-4) are defined by antigenic differences on the viral envelope protein, E, and together, they comprise a species within the Flavivirus genus of the Flaviviridae family. This family of small enveloped viruses with positive-sense RNA genomes encompasses other human pathogens, including West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and hepatitis C virus (HCV). A recent evidence-based study suggests that approximately 300 million DENV infections occur annually (1), and no vaccine or specific antiviral drug is currently available to treat it. DENV vaccine development is a major challenge due to the antibody-dependent enhancement of infection, a phenomenon in which neutralizing antibodies against one DENV serotype can exacerbate disease upon subsequent infection with another serotype (2, 3). A parallel exploration of antiviral strategies to combat DENV infection is therefore crucial.Resistance to antiviral drugs that act against viral targets occurs rapidly due to the intrinsically high mutation rate of RNA virus polymerases. Host-targeted antivirals that can complement these more traditional antivirals may make the acquisition of resistance to antiviral drugs much less likely and may also offer broad-spectrum activity against phylogenetically related viruses. The interactions between DENV and host lipid biosynthetic, metabolic, trafficking, and signal transducing pathways represent a rich and largely unexplored class of targets for host-targeted antiviral strategies. DENV and other RNA viruses rely entirely on host lipids to supply the membranes essential for the viral replication cycle, and the interaction of viruses with lipid-related processes in the host cell is highlighted by recent studies documenting specific perturbations of these pathways by viruses (4). In addition, so-called bioactive lipids can regulate cellular processes by modulating signal transduction cascades that may impinge on viral infection. Thus, small molecules that act on host-cell lipid signaling and metabolism are attractive as potential anti-DENV compounds.To pursue the strategy of targeting host lipid metabolic and signaling pathways important for DENV infection, we screened a panel of bioactive lipids and small-molecule inhibitors of lipid metabolism for activity against DENV. We chose a library enriched for compounds with known safety and bioavailability profiles to increase our likelihood of identifying clinically useful anti-DENV compounds. We present here the identification of the bioactive lipid 4-hydroxyphenyl retinamide (4-HPR) as an inhib-

The spermatogenesis associated 4 gene (Spata4, previously named TSARG2) was demonstrated to participate in spermatogenesis. Here we report that Spata4 is expressed in osteoblasts and that overexpression of Spata4 accelerates osteoblast differentiation and mineralization in MC3T3-E1 cells. We found that Spata4 interacts with p-Erk1/2 in the cytoplasm and that overexpression of Spata4 enhances the phosphorylation of Erk1/2. Intriguingly, we observed that Spata4 increases the transcriptional activity of Runx2, a critical transcription factor regulating osteoblast differentiation. We showed that Spata4-activated Runx2 is through the activation of Erk1/2. Consistent with this observation, we found that overexpression of Spata4 increases the expression of osteoblastic marker genes, including osteocalcin (Ocn), Bmp2, osteopontin (Opn), type 1 collagen, osterix (Osx), and Runx2. We concluded that Spata4 promotes osteoblast differentiation and mineralization through the Erk-activated Runx2 pathway. Our findings provided new evidence that Spata4 plays a role in regulation of osteoblast differentiation.