scholarly journals Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking

2016 ◽  
Vol 113 (10) ◽  
pp. 2756-2761 ◽  
Author(s):  
Stefania Militi ◽  
Elizabeth S. Maywood ◽  
Colby R. Sandate ◽  
Johanna E. Chesham ◽  
Alun R. Barnard ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Feedback Loop ◽  
Mutant Mouse ◽  
Protein Complexes ◽  
Causal Link ◽  
Structural Basis ◽  
Pas Domain ◽  
Period 2 ◽  
Clock Proteins ◽  
Functional Relevance

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1–CLOCK complexes is suppressed by PER–CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2Edo/Edo; Csnk1eTau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.

scholarly journals RNA-binding proteins and circadian rhythms in Arabidopsis thaliana

2001 ◽  
Vol 356 (1415) ◽  
pp. 1755-1759 ◽  
Author(s):  
Dorothee Staiger
Keyword(s):  
Arabidopsis Thaliana ◽  
Feedback Loop ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcriptional Level ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Circadian Oscillators ◽  
Clock Proteins ◽  
Reverse Genetic Approach

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA–binding protein At GRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: At GRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: At grp7 gene transcription is rhythmically activated by the circadian clock during the day. The At GPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, the At GRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the At GRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

scholarly journals Dissecting the conformation of glycans and their interactions with proteins

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Sheng-Hung Wang ◽  
Tsai-Jung Wu ◽  
Chien-Wei Lee ◽  
John Yu
Keyword(s):  
Conformational Changes ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Unmet Need ◽  
Protein Molecule ◽  
Structural Basis ◽  
Glycan Structure ◽  
Chronic Obstructive ◽  
Technology Platforms

Abstract The use of in silico strategies to develop the structural basis for a rational optimization of glycan-protein interactions remains a great challenge. This problem derives, in part, from the lack of technologies to quantitatively and qualitatively assess the complex assembling between a glycan and the targeted protein molecule. Since there is an unmet need for developing new sugar-targeted therapeutics, many investigators are searching for technology platforms to elucidate various types of molecular interactions within glycan-protein complexes and aid in the development of glycan-targeted therapies. Here we discuss three important technology platforms commonly used in the assessment of the complex assembly of glycosylated biomolecules, such as glycoproteins or glycosphingolipids: Biacore analysis, molecular docking, and molecular dynamics simulations. We will also discuss the structural investigation of glycosylated biomolecules, including conformational changes of glycans and their impact on molecular interactions within the glycan-protein complex. For glycoproteins, secreted protein acidic and rich in cysteine (SPARC), which is associated with various lung disorders, such as chronic obstructive pulmonary disease (COPD) and lung cancer, will be taken as an example showing that the core fucosylation of N-glycan in SPARC regulates protein-binding affinity with extracellular matrix collagen. For glycosphingolipids (GSLs), Globo H ceramide, an important tumor-associated GSL which is being actively investigated as a target for new cancer immunotherapies, will be used to demonstrate how glycan structure plays a significant role in enhancing angiogenesis in tumor microenvironments.

scholarly journals Structural basis for DEAH-helicase activation by G-patch proteins

2020 ◽  
Vol 117 (13) ◽  
pp. 7159-7170 ◽  
Author(s):  
Michael K. Studer ◽  
Lazar Ivanović ◽  
Marco E. Weber ◽  
Sabrina Marti ◽  
Stefanie Jonas
Keyword(s):  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Rna Helicases ◽  
Catalytic Core ◽  
Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

scholarly journals Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46

2017 ◽  
Vol 292 (18) ◽  
pp. 7462-7473 ◽  
Author(s):  
Michael Taschner ◽  
André Mourão ◽  
Mayanka Awasthi ◽  
Jerome Basquin ◽  
Esben Lorentzen
Keyword(s):  
Protein Complexes ◽  
Adaptor Protein ◽  
Intraflagellar Transport ◽  
Structural Basis ◽  
Fallopian Tubes ◽  
Large Protein ◽  
Terminal Domain ◽  
Unicellular Organisms ◽  
Motile Cilia ◽  
The Individual

Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

scholarly journals Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jennifer L Fribourgh ◽  
Ashutosh Srivastava ◽  
Colby R Sandate ◽  
Alicia K Michael ◽  
Peter L Hsu ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Feedback Loop ◽  
Pas Domain ◽  
Transactivation Domain ◽  
Transcriptional Repressors ◽  
Circadian Period ◽  
Circadian Timing ◽  
Homology Region ◽  
Differential Binding ◽  
Differential Affinity

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

scholarly journals New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gabriella Angiulli ◽  
Harveer Singh Dhupar ◽  
Hiroshi Suzuki ◽  
Irvinder Singh Wason ◽  
Franck Duong Van Hoa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Structural Basis ◽  
New Approach ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Membrane Protein Complexes ◽  
Membrane Protein Reconstitution

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

scholarly journals A model for the regulation of the timing of cell division by the circadian clock in the cyanobacterium Synechococcus elongatus

2019 ◽  
Author(s):  
Po-Yi Ho ◽  
Bruno M.C. Martins ◽  
Ariel Amir
Keyword(s):  
Circadian Clock ◽  
Time Window ◽  
Volume Growth ◽  
Synechococcus Elongatus ◽  
Clock Signal ◽  
Cell Divisions ◽  
Activity Peak ◽  
Period 2 ◽  
Clock Proteins ◽  
Over Time

1SummaryCells of the cyanobacterium Synechococcus elongatus possess a circadian clock in the form of three core clock proteins (the Kai proteins) whose concentrations and phosphorylation states oscillate with daily periodicity under constant conditions [1]. The circadian clock regulates the cell cycle such that the timing of cell divisions is biased towards certain times during the circadian period [2, 3, 4, 5], but the mechanism underlying how the clock regulates division timing remains unclear. Here, we propose a mechanism in which a protein limiting for division accumulates at a rate proportional to cell volume growth and modulated by the clock. This “modulated rates” model, in which the clock signal is integrated over time to affect division timing, differs fundamentally from the previously proposed “gating” concept, in which the clock is assumed to suppress divisions during a specific time window [2, 3]. We found that while both models can capture the single-cell statistics of division timing in S. elongatus, only the modulated rates model robustly places divisions away from darkness during changes in the environment. Moreover, within the framework of the modulated rates model, existing experiments on S. elongatus are consistent with the simple mechanism that division timing is regulated by the accumulation of a division limiting protein in phase with genes whose activity peak at dusk.

Human NLRP1: From the shadows to center stage

2021 ◽  
Vol 219 (1) ◽  
Author(s):  
Stefan Bauernfried ◽  
Veit Hornung
Keyword(s):  
Cell Death ◽  
Host Defense ◽  
Essential Role ◽  
Protein Complexes ◽  
Cell Damage ◽  
Specific Cell ◽  
Multimeric Protein ◽  
Functional Relevance ◽  
Molecular Mode ◽  
Pro Inflammatory Cytokines

In response to infection or cell damage, inflammasomes form intracellular multimeric protein complexes that play an essential role in host defense. Activation results in the maturation and subsequent secretion of pro-inflammatory cytokines of the IL-1 family and a specific cell death coined pyroptosis. Human NLRP1 was the first inflammasome-forming sensor identified at the beginning of the millennium. However, its functional relevance and its mechanism of activation have remained obscure for many years. Recent discoveries in the NLRP1 field have propelled our understanding of the functional relevance and molecular mode of action of this unique inflammasome sensor, which we will discuss in this perspective.

scholarly journals The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells

2014 ◽  
Vol 25 (19) ◽  
pp. 3081-3093 ◽  
Author(s):  
Tetsuya Gotoh ◽  
Marian Vila-Caballer ◽  
Carlo S. Santos ◽  
Jingjing Liu ◽  
Jianhua Yang ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Negative Regulator ◽  
Trimeric Complex ◽  
Physiological Processes ◽  
Period 2 ◽  
Damage Response ◽  
Stress Signals ◽  
Clock Mechanism

Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

Sign in / Sign up

Export Citation Format

Share Document