Single-Nucleus Landscape of Glial Cells and Neurons in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease with a projected significant increase in incidence. Therefore, this study analyzed single-nucleus AD data to provide a theoretical basis for the clinical development and treatment of AD. We downloaded AD-related monocyte data from the Gene Expression Omnibus database, annotated cells, compared cell abundance between groups, and investigated glial and neuronal cell biological processes and pathways through functional enrichment analysis.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework.

Discovery of an Orally Bioavailable and Selective PKMYT1 Inhibitor, RP-6306.

PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound was identified as a weak PKMYT1 inhibitor.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality.

A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency.

The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA.

Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment.

The human NDR family kinases control diverse aspects of cell growth, and are regulated through phosphorylation and association with scaffolds such as MOB1. Here, we report the crystal structure of the human NDR1 kinase domain in its non-phosphorylated state, revealing a fully resolved atypically long activation segment that blocks substrate binding and stabilizes a non-productive position of helix αC. Consistent with an auto-inhibitory function, mutations within the activation segment of NDR1 dramatically enhance in vitro kinase activity.

MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway.

The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ.

Regulation of Protein Interactions by ps ne inder (MOB1) Phosphorylation.

MOB1 is a multifunctional protein best characterized for its integrative role in regulating Hippo and NDR pathway signaling in metazoans and the Mitotic Exit Network in yeast. Human MOB1 binds both the upstream kinases MST1 and MST2 and the downstream AGC group kinases LATS1, LATS2, NDR1, and NDR2. Binding of MOB1 to MST1 and MST2 is mediated by its phosphopeptide-binding infrastructure, the specificity of which matches the phosphorylation consensus of MST1 and MST2.

Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity.

RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.

Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization.

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth.

Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system.

The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family.

Vac14 protein multimerization is a prerequisite step for Fab1 protein complex assembly and function.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P2 misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P2 whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction.

OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function.

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168.

Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine.

Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known.

"Unraveling the tail" of how SRPK1 phosphorylates ASF/SF2.

In this issue of Molecular Cell, Ngo et al. (2008) describe the crystal structure of the SRPK1 protein kinase in complex with its substrate, the spliceosome factor ASF/SF2, providing an unprecedented view of multiple targeting mechanisms in action on a single substrate.

CUL7 is a novel antiapoptotic oncogene.

Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53.

p53-Dependent transcriptional repression of c-myc is required for G1 cell cycle arrest.

The ability of p53 to promote apoptosis and cell cycle arrest is believed to be important for its tumor suppression function. Besides activating the expression of cell cycle arrest and proapoptotic genes, p53 also represses a number of genes. Previous studies have shown an association between p53 activation and down-regulation of c-myc expression.

A structure-based model of the c-Myc/Bin1 protein interaction shows alternative splicing of Bin1 and c-Myc phosphorylation are key binding determinants.

The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1.

Promoter-binding and repression of PDGFRB by c-Myc are separable activities.

The c-Myc transcription factor represses the mRNA expression of the platelet-derived growth factor receptor beta gene (PDGFRB). Using chromatin immunoprecipitation, we show that c-Myc binds to the proximal promoter of the PDGFRB gene in proliferating rat fibroblasts. Interestingly, mutant c-Myc proteins that are unable to repress PDGFRB gene expression, c-Myc(dBR) and c-Myc(d106-143), are still able to bind to the promoter in vivo.

c-Myc represses the proximal promoters of GADD45a and GADD153 by a post-RNA polymerase II recruitment mechanism.

The c-Myc cellular oncogene has diverse activities, including transformation, proliferation, and apoptosis. These activities are dependent on the ability of c-Myc to regulate gene transcription. c-Myc downregulates the GADD45a and GADD153 (DDTI3) genes that are induced in response to genotoxic stresses and that encode protein products with antiproliferative activities.

Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression.

The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo.

Functional analysis of the N-terminal domain of the Myc oncoprotein.

Myc is a multifunctional nuclear phosphoprotein that can drive cell cycle progression, apoptosis and cellular transformation. Myc orchestrates these activities at the molecular level by functioning as a regulator of gene transcription to activate or repress specific target genes. Previous studies have shown that both the Myc N-terminal domain (NTD) and the C-terminal domain (CTD) are essential for Myc functions.