Many proteins are glycosylated on Ser or Thr residues, but the mechanism of O-glycosylation is only poorly understood. A recent study by Chen et al. (Rapoport Lab, in collaboration with Paul Sullam’s lab at San Francisco Veteran Affairs Medical Center) reports on the mechanism by which the cytosolic O-glycosyltransferase GtfA/B of Streptococcus gordonii modifies the Ser/Thr-rich repeats of adhesin, a protein that mediates the attachment of the bacterium to host cells. Crystal structures and biochemical experiments indicate that, during a first phase of glycosylation, the conformation of GtfB is restrained by GtfA to bind substrate with unmodified Ser/Thr residues. In a slow second phase, GtfB recognizes residues that are already modified with N-acetylglucosamine by converting into a relaxed conformation. These results explain how the glycosyltransferase modifies a progressively changing substrate molecule.
Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of V(D)J gene segments are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase precisely recognizes and cleaves two different recombination signal sequences (12-RSS and 23-RSS), and forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. As described in a recent paper in Cell, the Liao Lab (in collaboration with Dr. Hao Wu's lab at Boston Children's Hospital) used single-particle cryo-electron microscopy (cryo-EM) to determine the structures of synaptic RAG1-RAG2 complexes at up to 3.4 Å resolution. These structures reveal an RSS-induced closed conformation that activates catalysis and uncovers the molecular basis for the 12/23 rule.
This new work, by Katrin Svensson in the Spiegelman Lab and recently published in Cell Metabolism, has found a new function for Slit2, a protein secreted from beige cells. Slit2-C, a C-terminal cleaved fragment of Slit2 circulates in plasma and improves glucose homeostasis in mice by activating a thermogenic gene expression program and the classical PKA signaling pathway in fat cells. These findings establish a previously unknown peripheral role for a Slit2 fragment that has therapeutic potential for the treatment of diabetes and other metabolic disorders.
An international team led by researchers at Harvard Medical School and Massachusetts General Hospital has devised a new way to approach the problem of multidrug-resistant fungal infections that can be life-threatening to people with weakened immune systems. The Näär Lab and Gerhard Wagner's lab identified this compound from a library of 150,000 small molecules. To learn more, see here.
RPB Stein Innovation Awards provide funds to researchers in the ophthalmology department and to basic science or other relevant vision researchers outside of the ophthalmology department (but within the institution) with a common goal of understanding the visual system and the diseases that compromise its function. These awards are intended to provide seed money to proposed high-risk/high-gain vision science research which is innovative, cutting-edge, and demonstrates out-of-the-box thinking.
Congrats to Anders!
The epidemic of obesity and diabetes have increased interest in pathways that cause an increase in metabolic rates. The classical pathway of adaptive thermogenesis involves the function of mitochondrial Uncoupling Protein 1 (UCP1) in brown fat. This new work by the Spiegelman lab and published recently in Cell, shows that beige fat cells have another pathway used to generate heat and dissipate chemical energy. Their mitochondria utilize a futile cycle of creatine phosphorylation/dephosphorylation to elevate respiration and expend calories as heat. This new pathway may offer new insights into thermal regulation and offer new targets for human metabolic disorders.
NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, the Harper lab demonstrated that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting.
Mancias, JD, Pontano Vaites L, Nissim S, Biancur DE, Kim AJ, Wang X, Liu Y, Goessling W, Kimmelman AC, Harper JW. (2015) Ferritinophagy via NCOA4 is required for erythroid development and is regulated by an iron dependent HERC2-mediated proteolysis. eLife
The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to ‘segregate’ ubiquitylated proteins from their binding partners, and has been implicated in numerous pathways ranging from ERAD to repair of damaged DNA. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells reported in Nature Cell Biology, the Harper lab revealed a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways based on Gene Ontology analysis. Through detailed analysis of the interaction partners for the unstudied adaptor UBXN10, they uncover a role for this adaptor and VCP/p97 in ciliogenesis. UBXN10 associates with the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. Using TIRF microscopy in living cells, the demonstrate that UBXN10 is localized in cilia and traffics in cilia with the IFT-B complex. Moreover, deletion of UBXN10 using gene-editing renders cells unable to form cilia. Pharmacological inhibition of VCP destabilized they IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left–right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP–UBXN10 in ciliogenesis.
Raman, M, Sergeev, M, Garnaas M, Lydeard JR, Huttlin EL, Goessling W, Shah JV, and Harper JW. (2015) Systematic proteomics of the VCP–UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis. Nature Cell Biology
Previous work has identified roles for the protein kinase PINK1 and the ubiquitin ligase PARKIN in the synthesis of ubiquitin chains on the surface of mitochondria in response to mitochondrial damage. But how these ubiquitin chains target damaged mitochondria to autophagosomes for “mitophagy” and the regulation of this process remained unclear. New work from the Harper Lab, published in Molecular Cell, defines a mechanism in which ubiquitin chains on mitochondria recruit 4 autophagy adaptors (OPTN, NDP52, p62, and TAX1BP1) through their ubiquitin binding domains to specific foci on damaged mitochondria. This recruitment leads to activation of the TBK1 kinase, which associates with OPTN, NDP52, and p62. In a concerted fashion, TBK1 phosphorylates OPTN, NDP52, and p62. Biochemical studies demonstrated that phosphorylation of OPTN by TBK1 further stimulates its intrinsic ubiquitin binding activity. Thus, this mechanism provides a self-reinforcing system that links these ubiquitin binding autophagy receptors with damaged mitochondria. Importantly, recent work has demonstrated that both TBK1 and OPTN are mutated in ALS. Thus, this work demonstrates that genes mutated in Parkinson’s Disease and ALS function in a common signal transduction pathway to help rid the cell of damaged mitochondria, and suggests that mechanisms for disposal of damaged mitochondria and possibly other toxic cellular components may be commonly targeted in such neurodegenerative diseases.
The Pex1 and Pex6 ATPases participate in peroxisome biogenesis. Mutations affecting these proteins are the most common cause of various generally fatal peroxisomal diseases. In a collaboration among the laboratories of Rapoport, Walz, and DiMaio/Baker, structures of the Pex1/Pex6 complex were determined in the presence of different nucleotides by cryo-electron microscopy (Blok et al., 2015). The structures support the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to that of another ATPase, p97, in ER-associated protein degradation.