Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells [DNA and Chromosomes]

April 23rd, 2018 by Jun Wu, Pengcheng Wang, Lin Li, Changjun You, Yinsheng Wang

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrated the roles of TLS polymerases in replicative bypass of these lesions in human cells.

Effects of hypo O-GlcNAcylation on Drosophila development [Developmental Biology]

March 27th, 2018 by Daniel N Mariappa, Andrew Ferenbach, Daan M.F. van Aalten

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the polycomb gene O-GlcNAc transferase (OGT, also known as super sex combs [sxc]) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxcK872M, was recessive lethal, whereas a second mutant, the hypomorphic sxcH537A, was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxcH537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxcH537A and a null allele of Drosophila host cell factor (dHcf), encoding an extensively O-GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxcH537A flies lacking a copy of skuld (skd), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxcH537A enabled us to identify pleiotropic effects of globally reduced protein O-GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development.

Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef [Microbiology]

March 27th, 2018 by Amanda J. Chase, Rebecka Wombacher, Oliver T. Fackler

The HIV-1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network (TGN), while the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18 amino acid SH4 domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for TGN retargeting by Nef, as well as the importance of the amino acid at position 8 for determining Nef-sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone (LPC) Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that (i) analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and (ii) intrinsic properties define SH4 domains as cargo of this Nef-sensitive LPC-GTPase transport cycle.
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Phospholipid Subcellular Localization and Dynamics [Membrane Biology]

March 27th, 2018 by Yanbo Yang, Minhyoung Lee, Gregory D. Fairn

Membrane biology seeks to understand how lipids and proteins within bilayers assemble into large structures such as organelles and the plasma membrane. Historically, lipids were thought to merely provide structural support for bilayer formation and membrane protein function. Research has now revealed that phospholipid metabolism regulates nearly all cellular processes. Sophisticated techniques helped identify >10,000 lipid species, suggesting that lipids support many biological processes. Here, we highlight the synthesis of the most abundant glycerophospholipids classes and their distribution in organelles. We review vesicular and nonvesicular transport pathways shuttling lipids between organelles and discuss lipid regulators of membrane trafficking and second messengers in eukaryotic cells.

Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by {alpha}-amanitin [Protein Structure and Folding]

March 17th, 2018 by Xiangyang Liu, Lucas Farnung, Christoph Wigge, Patrick Cramer

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II, but forms additional contacts with metazoan-specific residues, which explain why its affinity to mammalian Pol II is ~3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.

The major facilitator transporter Str3 is required for low-affinity heme acquisition in Schizosaccharomyces pombe [Microbiology]

March 16th, 2018 by Vincent Normant, Thierry Mourer, Simon Labbe

In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-μM concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, a Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530Y-X3-Y534 and 552S-X4-Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 μM. Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.

Ubiquitin-conjugating enzyme E2 D1 (Ube2D1) mediates lysine-independent ubiquitination of the E3 ubiquitin ligase March-I [Protein Synthesis and Degradation]

February 1st, 2018 by Lei Lei, Joanna Bandola-Simon, Paul A. Roche

March-I is a membrane-bound E3 ubiquitin ligase belonging to the membrane-associated RING-CH (March) family. March-I ubiquitinates and down-regulates expression of major histocompatibility complex (MHC) class II and cluster of differentiation 86 (CD86) in antigen presenting cells. March-I expression is regulated both transcriptionally and post-translationally and it has been reported that the March-I is ubiquitinated and that this ubiquitination contributes to March-I turnover. However, the molecular mechanism regulating March-I ubiquitination and the importance of March-I's E3 ligase activity for March-I ubiquitination are not fully understood. Here we confirmed that although March-I is ubiquitinated, it is not ubiquitinated on a lysine residue as a lysine-less March-I variant was ubiquitinated similarly to wild-type March-I. We found that March-I E3 ligase activity is not required for its ubiquitination and does not regulate March-I protein expression, suggesting that March-I does not undergo autoubiquitination. Knocking down ubiquitin-conjugating enzyme E2 D1 (Ube2D1) impaired March-I ubiquitination, increased March-I expression, and enhanced March-I-dependent downregulation of MHC class II proteins. Taken together, our results suggest that March-I undergoes lysine-independent ubiquitination by an as yet unidentified E3 ubiquitin ligase that together with Ube2D1 regulates March-I expression.
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Leptin enhances hypothalamic LDHA-dependent glucose sensing to lower glucose production in high-fat fed rats. [Metabolism]

January 26th, 2018 by Mona A. Abraham, Mozhgan Rasti, Paige V. Bauer, Tony K.T. Lam

The responsiveness of glucose sensing per se to regulate whole-body glucose homeostasis is dependent on the ability of a rise in glucose to lower hepatic glucose production and increase peripheral glucose uptake in vivo. In both rodents and humans, glucose sensing is lost in diabetes and obesity but the site(s) of impairment remain elusive. We here first report that short-term high-fat feeding dirsupts hypothalamic glucose sensing to lower glucose production in rats. Second, leptin administration into the hypothalamus of high-fat fed rats restored hypothalamic glucose sensing to lower glucose production during a pancreatic (basal insulin)-euglycemic clamp and increased whole-body glucose tolerance during an intravenous glucose tolerance test. Finally, both chemical inhibition of hypothalamic lactate dehydrogenase (LDH) (achieved via hypothalamic LDH inhibitor oxamate infusion) and molecular knockdown of LDHA (achieved via hypothalamic lentiviral-LDHA shRNA injection) negated the ability of hypothalamic leptin infusion to enhance glucose sensing to lower glucose production in high-fat fed rats. In summary, our findings illustrate that leptin enhances LDH-A-dependent glucose sesning in the hypothalamus to lower glucose production in high-fat fed rodents in vivo.

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species [Molecular Biophysics]

January 25th, 2018 by D. Fernando Estrada

Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH)2D3. Its function relies on formation of a P450–redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx–CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450–Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1­–Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH)2D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge–neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.

Transcriptional and post-transcriptional regulation of autophagy in the yeast Saccharomyces cerevisiae [Cell Biology]

January 25th, 2018 by Elizabeth Delorme-Axford, Daniel J. Klionsky

Autophagy is a highly conserved catabolic pathway that is vital for development, cell survival and the degradation of dysfunctional organelles and toxic aggregates. Dysregulation of autophagy is associated with cancer, neurodegeneration and lysosomal storage diseases. Accordingly, autophagy is precisely regulated at multiple levels (transcriptional, post-transcriptional, epigenetic, translational and post-translational) to prevent aberrant activity. Various model organisms exist to study autophagy but the baker's yeast Saccharomyces cerevisiae continues to be very advantageous for genetic and biochemical analysis of non-selective and selective autophagy. In this review, we focus on the cellular mechanisms that regulate autophagy transcriptionally and post-transcriptionally in S. cerevisiae.