Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

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Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613374114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3699-E3708 ◽

Cited By ~ 34

Author(s):

Daisuke Ono ◽

Sato Honma ◽

Yoshihiro Nakajima ◽

Shigeru Kuroda ◽

Ryosuke Enoki ◽

...

Keyword(s):

Circadian Rhythms ◽

Suprachiasmatic Nucleus ◽

Clock Genes ◽

Behavioral Rhythm ◽

Calcium Indicator ◽

Intracellular Ca ◽

Regional Specificity ◽

Free Running ◽

Activity Onset ◽

And Behavior

The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.

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The cohesin release factor Wapl interacts with Bub3 to govern SAC activity in female meiosis I

Science Advances ◽

10.1126/sciadv.aax3969 ◽

2020 ◽

Vol 6 (15) ◽

pp. eaax3969

Author(s):

Changyin Zhou ◽

Yilong Miao ◽

Zhaokang Cui ◽

Xiayan ShiYang ◽

Yu Zhang ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Mass Spectrometry Analysis ◽

Female Meiosis ◽

Spectrometry Analysis ◽

Binding Partner ◽

Meiotic Progression ◽

Cohesin Subunit ◽

Normal Meiosis ◽

Meiosis I

During mitotic prophase, cohesins are removed from chromosome arms by Wapl to ensure faithful sister chromatid separation. However, during female meiosis I, the resolution of chiasmata requires the proteolytic cleavage of cohesin subunit Rec8 along chromosome arms by Separase to separate homologs, and thus the role of Wapl remained unknown. Here, we report that Wapl functions as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Depletion of Wapl accelerates meiotic progression, inactivates SAC, and causes meiotic defects such as aberrant spindle/chromosome structure and incorrect kinetochore-microtubule (K-MT) attachment, consequently leading to aneuploid eggs. Notably, we identify Bub3 as a binding partner of Wapl by immunoprecipitation and mass spectrometry analysis. We further determine that Wapl controls the SAC activity by maintaining Bub3 protein level and document that exogenous Bub3 restores the normal meiosis in Wapl-depleted oocytes. Together, our findings uncover unique, noncanonical roles for Wapl in mediating control of the SAC in female meiosis I.

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Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

Scientific Reports ◽

10.1038/s41598-021-00045-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Victoria Palin ◽

Matthew Russell ◽

Robert Graham ◽

John D. Aplin ◽

Melissa Westwood

Keyword(s):

Fetal Growth ◽

Protein Function ◽

Molecular Mechanisms ◽

Nutrient Sensing ◽

Mass Spectrometry Analysis ◽

Hexosamine Biosynthetic Pathway ◽

Spectrometry Analysis ◽

Increased Risk ◽

Associated Proteins ◽

Regulate Protein

AbstractWomen with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.

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The SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

10.1101/2021.05.09.443327 ◽

2021 ◽

Author(s):

Sofya A Polyanskaya ◽

Rosamaria Y Moreno ◽

Bin Lu ◽

Ruopeng Feng ◽

Yu Yao ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mass Spectrometry Analysis ◽

Phosphatase Domain ◽

Spectrometry Analysis ◽

Signaling Complex ◽

Catalytic Function ◽

Binding Partners ◽

Catalytic Removal ◽

Acute Myeloid

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we used domain-focused CRISPR screening to identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

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Spontaneous Recovery of Circadian Organization in Mice Lacking a Core Component of the Molecular Clockwork

Journal of Biological Rhythms ◽

10.1177/07487304211060896 ◽

2021 ◽

pp. 074873042110608

Author(s):

Jonathan P. Riggle ◽

Kenneth G. Onishi ◽

Jharnae A. Love ◽

Dana E. Beach ◽

Irving Zucker ◽

...

Keyword(s):

Circadian Rhythms ◽

Spontaneous Recovery ◽

Clock Genes ◽

Feedback Loops ◽

Constant Darkness ◽

Core Component ◽

Period 2 ◽

Circadian Organization ◽

Circadian Clock Genes ◽

Free Running

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene ( Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

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Deubiquitinating enzyme USP9X regulates cellular clock function by modulating the ubiquitination and degradation of a core circadian protein BMAL1

Biochemical Journal ◽

10.1042/bcj20180005 ◽

2018 ◽

Vol 475 (8) ◽

pp. 1507-1522 ◽

Cited By ~ 7

Author(s):

Yang Zhang ◽

Chunyan Duan ◽

Jing Yang ◽

Suping Chen ◽

Qing Liu ◽

...

Keyword(s):

Circadian Rhythm ◽

Circadian Clock ◽

Clock Genes ◽

Affinity Purification ◽

Mass Spectrometry Analysis ◽

Regulatory Function ◽

Spectrometry Analysis ◽

The Core ◽

Clock Proteins ◽

Clock Protein

Living organisms on the earth maintain a roughly 24 h circadian rhythm, which is regulated by circadian clock genes and their protein products. Post-translational modifications of core clock proteins could affect the circadian behavior. Although ubiquitination of core clock proteins was studied extensively, the reverse process, deubiquitination, has only begun to unfold and the role of this regulation on circadian function is not completely understood. Here, we use affinity purification and mass spectrometry analysis to identify probable ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) as an interacting protein of the core clock protein aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1). Through biochemical experiments, we discover that USP9X reduces BMAL1 ubiquitination, enhances its stability, and increases its protein level, leading to the elevated transcriptional activity. Bioluminescence measurement reveals that USP9X knockdown decreases the amplitude of the cellular circadian rhythm but the period and phase are not affected. Our experiments find a new regulator for circadian clock at the post-translational level and demonstrate a different regulatory function for the circadian clock through the deubiquitination and the up-regulation of the core clock protein BMAL1 in the positive limb of the transcription–translation feedback loop.

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Period2 3′-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706611114 ◽

2017 ◽

Vol 114 (42) ◽

pp. E8855-E8864 ◽

Cited By ~ 33

Author(s):

Seung-Hee Yoo ◽

Shihoko Kojima ◽

Kazuhiro Shimomura ◽

Nobuya Koike ◽

Ethan D. Buhr ◽

...

Keyword(s):

Circadian Rhythms ◽

Light Pulse ◽

Protein Level ◽

Temperature Compensation ◽

Clock Genes ◽

Protein Translation ◽

Protein Accumulation ◽

Free Running ◽

Protein Oscillation

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3′-UTR region: Per2::Luc, which retains the endogenous Per2 3′-UTR and Per2::LucSV, where the endogenous Per2 3′-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3′-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc. Analysis of the Per2 3′-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3′-UTR, miR-24, and PER2 in Per2 expression and core clock function.

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Phototropin Interactions with SUMO Proteins

Plant and Cell Physiology ◽

10.1093/pcp/pcab027 ◽

2021 ◽

Author(s):

Justyna Łabuz ◽

Olga Sztatelman ◽

Dominika Jagiełło-Flasińska ◽

Paweł Hermanowicz ◽

Aneta Bażant ◽

...

Keyword(s):

Blue Light ◽

Covalent Modification ◽

Mass Spectrometry Analysis ◽

Protein Abundance ◽

Growth Responses ◽

Yeast Two Hybrid ◽

Spectrometry Analysis ◽

Mature Leaves ◽

The Stability ◽

Two Hybrid

Abstract The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.

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