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Summary: RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here, we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response. [ABSTRACT FROM AUTHOR]

Large G protein α subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Gα subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein–protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Gα subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling. [Copyright &y& Elsevier]

Summary Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing. [ABSTRACT FROM AUTHOR]

Summary The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease. [ABSTRACT FROM AUTHOR]

Summary: Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance. [ABSTRACT FROM AUTHOR]

Summary: The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism. [Copyright &y& Elsevier]

Idiopathic pulmonary fibrosis (IPF) is a lethal scarring lung disease that affects older adults. Heterozygous rare mutations in the genes encoding telomerase are found in ∼15% of familial cases. We have used linkage to map another disease-causing gene in a large family with IPF and adenocarcinoma of the lung to a 15.7 Mb region on chromosome 10. We identified a rare missense mutation in a candidate gene, SFTPA2, within the interval encoding surfactant protein A2 (SP-A2). Another rare mutation in SFTPA2 was identified in another family with IPF and lung cancer. Both mutations involve invariant residues in the highly conserved carbohydrate-recognition domain of the protein and are predicted to disrupt protein structure. Recombinant proteins carrying these mutations are retained in the endoplasmic reticulum and are not secreted. These data are consistent with SFTPA2 germline mutations that interfere with protein trafficking and cause familial IPF and lung cancer. [ABSTRACT FROM AUTHOR]

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy. [ABSTRACT FROM AUTHOR]

RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development. • Validation of compound B as a specific RUVBL1/2 ATPase inhibitor • RUVBL1/2 ATPase activity is required for PAQosome, not INO80-family, complexes • NSCLC requires RUVBL1/2 ATPase activity for DNA replication • Compound B radiosensitizes NSCLC, but not normal lung epithelial cells Yenerall et al. identified a specific inhibitor of RUVBL1/2 ATPase activity, compound B, and demonstrate that RUVBL1/2 ATPase activity is required for PAQosome maturation/dissociation. Compound B kills non-small cell lung cancer (NSCLC) by inhibiting DNA replication. In addition, compound B radiosensitizes NSCLC, but not normal cells, an attractive property for future development. [ABSTRACT FROM AUTHOR]

Summary Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities. Graphical Abstract Highlights • SelO adopts a protein kinase fold with ATP flipped in the active site • SelO transfers AMP to Ser, Thr, and Tyr residues on protein substrates (AMPylation) • SelO AMPylates proteins involved in redox homeostasis • SelO protects cells from oxidative stress and regulates protein glutathionylation The structure of SelO, a conserved pseudokinase, reveals ATP flipped in the substrate binding pocket, leading to the discovery that SelO is actually an AMPylating enzyme. [ABSTRACT FROM AUTHOR]

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway., Competing Interests: Declaration of Interests R.E.C., A.S.L., R.B., and A.A. are employees and shareholders of Puma Biotechnology, Inc. A.G.Z. has received research and travel grants from Pfizer. A.B.H. receives research grant support from Takeda and has received a travel grant from Puma Biotechnology, Inc. C.L.A receives or has received research grants from Puma Biotechnology, Pfizer, Lilly, Bayer, Takeda, and Radius; holds stock options in Provista and Y-TRAP; serves or has served in an advisory role to Novartis, Merck, Lilly, Symphogen, Daiichi Sankyo, Radius, Taiho Oncology, H3Biomedicine, OrigiMed, Puma Biotechnology, and Sanofi; and reports scientific advisory board remuneration from the Komen Foundation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fumarate hydratase (FH) is a tumor suppressor, but how it acts is unclear. Two reports in this issue of Cancer Cell reveal that FH deficiency leads to succination of Keap1, stabilization of Nrf2, and induction of stress-response genes including HMOX1, which is important for the survival of FH-deficient cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy.