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.
  • Posted in Journal of Biological Chemistry, Publications
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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.

A Molecular Perspective of Mammalian Autophagosome Biogenesis [Membrane Biology]

January 25th, 2018 by Thomas J. Mercer, Andrea Gubas, Sharon A. Tooze

Autophagy is a highly conserved process, essential for the maintenance of cellular homeostasis. Autophagy occurs at a basal level in all cells, but can be upregulated during stress, starvation, or infection. Misregulation of autophagy has been linked to various disorders, including cancer, neurodegeneration and immune diseases. Here, we discuss the essential proteins acting in the formation of an autophagosome, with a focus on the ULK and VPS34 kinase complexes, PI3P effector proteins and the transmembrane autophagy-related protein ATG9. The function and regulation of these and other autophagy-related proteins, acting during formation will be addressed, in particular, during amino acid starvation.

Toxoplasma gondii disrupts {beta}1 integrin signaling and focal adhesion formation during monocyte hypermotility [Immunology]

January 2nd, 2018 by Joshua H. Cook, Norikiyo Ueno, Melissa B. Lodoen

The motility of blood monocytes is orchestrated by the activity of cell surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We have investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii. Compared to uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with LPS or E. coli. Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Y397) and Y925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Y402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing eGFP-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of eGFP-FAK-containing clusters over time compared to uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii- induced monocyte hypermotility.

The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane [Membrane Biology]

December 24th, 2017 by Katumori Segawa, Sachiko Kurata, Shigekazu Nagata

Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidyl- ethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a hetero-complex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C’s flippase activity. These residues were all located in CDC50A’s extracellular domain and were evolutionarily well conserved. Most of the mutations decreased CDC50A’s ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C’s PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A’s ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C’s ATP hydrolysis–coupled flippase activity.
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Chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone [Metabolism]

December 15th, 2017 by Kumsal A Tekirdag, Ana Maria Cuervo

A variety of mechanisms deliver cytosolic materials to the lysosomal compartment for degradation through autophagy. Here, we focus on two autophagic pathways, chaperone-mediated autophagy and endosomal microautophagy that rely on the cytosolic chaperone hsc70 for substrate targeting. Although hsc70 participates in triage of proteins for degradation by different proteolytic systems, the common characteristic shared by these two forms of autophagy, is that hsc70 binds directly to a specific five amino acids motif in the cargo protein for its autophagic targeting. We summarize the current understanding of the molecular machineries behind each of these types of autophagy.