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Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03–176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03–176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

Chemically induced protein degradation is a powerful strategy for perturbing cellular biochemistry. The predominant mechanism of action for protein degrader drugs involves induced proximity between the cellular ubiquitin conjugation machinery and the target. Unlike traditional small molecule enzyme inhibition, targeted protein degradation can clear an undesired protein from cells. We demonstrate here the use of peptide ligands for Kelch-Like Homology Domain Containing protein 2 (KLHDC2), a substrate adaptor protein and member of the cullin-2 (CUL2) ubiquitin ligase complex, for targeted protein degradation. Peptide-based bivalent compounds that can induce proximity between KLHDC2 and target proteins cause degradation of the targeted factors. The cellular activity of these compounds depends on KLHDC2 binding. This work demonstrates the utility of KLHDC2 for targeted protein degradation and exemplifies a strategy for the rational design of new peptide-based ligands useful for this purpose.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro 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 Mpro bound to two viral substrate peptides. The structures show how Mpro 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 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

Kinetochores control eukaryotic chromosome segregation by connecting chromosomal centromeres to spindle microtubules. Duplication of centromeric DNA necessitates kinetochore disassembly and subsequent reassembly on the nascent sisters. To search for a regulatory mechanism that controls the earliest steps of kinetochore assembly, we studied Mif2/CENP-C, an essential basal component. We found that Polo-like kinase (Cdc5) and Dbf4-dependent kinase (DDK) phosphorylate the conserved PEST region of Mif2/CENP-C and that this phosphorylation directs inner kinetochore assembly. Mif2 phosphorylation promotes kinetochore assembly in a reconstituted biochemical system, and it strengthens Mif2 localization at centromeres in cells. Disrupting one or more phosphorylation sites in the Mif2-PEST region progressively impairs cellular fitness and sensitizes cells to microtubule poisons. The most severe Mif2-PEST mutations are lethal in cells lacking otherwise non-essential Ctf19 complex factors. These data suggest that multi-site phosphorylation of Mif2/CENP-C is a robust switch that controls inner kinetochore assembly, ensuring accurate chromosome segregation.

SUMO homeostasis promotes error-free chromosome segregation. Quan et al. report the structure of a targeting peptide of the Ulp2 desumoylase bound to yeast Ctf3/CENP-I. Disrupting the interaction produces hyper-sumoylated kinetochores, demonstrating the existence of a conserved and dedicated pathway for the regulation of kinetochore sumoylation., The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

Kinetochore molecular architecture exemplifies "form follows function." The simplifications that generated the one-chromosome:one-microtubule linkage in point-centromere yeast have enabled strategies for systematic structural analysis and high-resolution visualization of many kinetochore components, leading to specific proposals for molecular mechanisms. We describe here some structural features that allow a kinetochore to remain attached to the end of a depolymerizing microtubule (MT) and some characteristics of the connections between substructures that permit very sensitive regulation by differential kinase activities. We emphasize in particular the importance of flexible connections between rod-like structural members and the integration of these members into a compliant cage-like assembly anchored on the MT by a sliding molecular ring.

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.

During a single human lifetime, nearly one quintillion chromosomes separate from their sisters and transit to their destinations in daughter cells. Unlike DNA replication, chromosome segregation has no template, and, unlike transcription, errors frequently lead to a total loss of cell viability. Rapid progress in recent years has shown how kinetochores enable faithful execution of this process by connecting chromosomal DNA to microtubules. These findings have transformed our idea of kinetochores from cytological features to immense molecular machines and now allow molecular interpretation of many long-appreciated kinetochore functions. In this review we trace kinetochore protein connectivity from chromosomal DNA to microtubules, relating new findings to important points of regulation and function.

The 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.

SummaryAccurate 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.

Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae , including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan- Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

PFMG5 (Pinctada fucata mantle gene 5) was identified from mantle tissue of the pearl oyster, Pinctada fucada (P. fucada). Here we report that PFMG5 decreased osteoblast differentiation marker alkaline phosphatase (ALP) activity and the transcript levels of osteoblast differentiation specific marker genes in MC3T3-E1 cells. PFMG5 was identified as a new molecule inhibiting osteoblast differentiation.

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. © 2011 American Society for Bone and Mineral Research