Dissecting the biophysics and biology of intrinsically disordered proteins.

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.

Circadian control of heparan sulfate levels times phagocytosis of amyloid beta aggregates.

Alzheimer's Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (Aß). AD has a bi-directional relationship with circadian disruption (CD) with sleep disturbances starting years before disease onset. However, the molecular mechanism underlying the relationship of CD and AD has not been elucidated. Myeloid-based phagocytosis, a key component in the metabolism of Aß, is circadianly-regulated, presenting a potential link between CD and AD. In this work, we revealed that the phagocytosis of Aß42 undergoes a daily circadian oscillation. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of Aß and that both HSPG binding and aggregation may play a role in this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD.

Post-transcriptional circadian regulation in macrophages organizes temporally distinct immunometabolic states.

Our core timekeeping mechanism, the circadian clock, plays a vital role in immunity. Although the mechanics of circadian control over the immune response is generally explained by transcriptional activation or repression derived from this clock's transcription-translation negative-feedback loop, research suggests that some regulation occurs beyond transcriptional activity. We comprehensively profiled the transcriptome and proteome of murine bone marrow-derived macrophages and found that only 15% of the circadian proteome had corresponding oscillating mRNA, suggesting post-transcriptional regulation influences macrophage clock regulatory output to a greater extent than any other tissue previously profiled. This regulation may be explained by the robust temporal enrichment we identified for proteins involved in degradation and translation. Extensive post-transcriptional temporal-gating of metabolic pathways was also observed and further corresponded with daily variations in ATP production, mitochondrial morphology, and phagocytosis. The disruption of this circadian post-transcriptional metabolic regulation impaired immune functionality. Our results demonstrate that cell-intrinsic post-transcriptional regulation is a primary driver of circadian output in macrophages and that this regulation, particularly of metabolic pathways, plays an important role in determining their response to immune stimuli.

MOSAIC: a joint modeling methodology for combined circadian and non-circadian analysis of multi-omics data.

MOTIVATION: Circadian rhythms are approximately 24-h endogenous cycles that control many biological functions. To identify these rhythms, biological samples are taken over circadian time and analyzed using a single omics type, such as transcriptomics or proteomics. By comparing data from these single omics approaches, it has been shown that transcriptional rhythms are not necessarily conserved at the protein level, implying extensive circadian post-transcriptional regulation. However, as proteomics methods are known to be noisier than transcriptomic methods, this suggests that previously identified arrhythmic proteins with rhythmic transcripts could have been missed due to noise and may not be due to post-transcriptional regulation. RESULTS: To determine if one can use information from less-noisy transcriptomic data to inform rhythms in more-noisy proteomic data, and thus more accurately identify rhythms in the proteome, we have created the Multi-Omics Selection with Amplitude Independent Criteria (MOSAIC) application. MOSAIC combines model selection and joint modeling of multiple omics types to recover significant circadian and non-circadian trends. Using both synthetic data and proteomic data from Neurospora crassa, we showed that MOSAIC accurately recovers circadian rhythms at higher rates in not only the proteome but the transcriptome as well, outperforming existing methods for rhythm identification. In addition, by quantifying non-circadian trends in addition to circadian trends in data, our methodology allowed for the recognition of the diversity of circadian regulation as compared to non-circadian regulation. AVAILABILITY AND IMPLEMENTATION: MOSAIC's full interface is available at https://github.com/delosh653/MOSAIC. An R package for this functionality, mosaic.find, can be downloaded at https://CRAN.R-project.org/package=mosaic.find. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ECHO: an application for detection and analysis of oscillators identifies metabolic regulation on genome-wide circadian output.

MOTIVATION: Time courses utilizing genome scale data are a common approach to identifying the biological pathways that are controlled by the circadian clock, an important regulator of organismal fitness. However, the methods used to detect circadian oscillations in these datasets are not able to accommodate changes in the amplitude of the oscillations over time, leading to an underestimation of the impact of the clock on biological systems. RESULTS: We have created a program to efficaciously identify oscillations in large-scale datasets, called the Extended Circadian Harmonic Oscillator application, or ECHO. ECHO utilizes an extended solution of the fixed amplitude oscillator that incorporates the amplitude change coefficient. Employing synthetic datasets, we determined that ECHO outperforms existing methods in detecting rhythms with decreasing oscillation amplitudes and in recovering phase shift. Rhythms with changing amplitudes identified from published biological datasets revealed distinct functions from those oscillations that were harmonic, suggesting purposeful biologic regulation to create this subtype of circadian rhythms. AVAILABILITY AND IMPLEMENTATION: ECHO's full interface is available at https://github.com/delosh653/ECHO. An R package for this functionality, echo.find, can be downloaded at https://CRAN.R-project.org/package=echo.find. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Conserved RNA helicase FRH acts nonenzymatically to support the intrinsically disordered neurospora clock protein FRQ.

Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed intrinsically disordered proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein FREQUENCY (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to FREQUENCY-Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via a pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

Circadian Oscillators: Around the Transcription-Translation Feedback Loop and on to Output.

From cyanobacteria to mammals, organisms have evolved timing mechanisms to adapt to environmental changes in order to optimize survival and improve fitness. To anticipate these regular daily cycles, many organisms manifest ∼24h cell-autonomous oscillations that are sustained by transcription-translation-based or post-transcriptional negative-feedback loops that control a wide range of biological processes. With an eye to identifying emerging common themes among cyanobacterial, fungal, and animal clocks, some major recent developments in the understanding of the mechanisms that regulate these oscillators and their output are discussed. These include roles for antisense transcription, intrinsically disordered proteins, codon bias in clock genes, and a more focused discussion of post-transcriptional and translational regulation as a part of both the oscillator and output.

Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock.

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module.

Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit.

INTRODUCTION: The circadian circuit, a roughly 24 h molecular feedback loop, or clock, is conserved from bacteria to animals and allows for enhanced organismal survival by facilitating the anticipation of the day/night cycle. With circadian regulation reportedly impacting as high as 80% of protein coding genes in higher eukaryotes, the protein-based circadian clock broadly regulates physiology and behavior. Due to the extensive interconnection between the clock and other cellular systems, chronic disruption of these molecular rhythms leads to a decrease in organismal fitness as well as an increase of disease rates in humans. Importantly, recent research has demonstrated that proteins comprising the circadian clock network display a significant amount of intrinsic disorder. MAIN BODY: In this work, we focus on the extent of intrinsic disorder in the circadian clock and its potential mechanistic role in circadian timing. We highlight the conservation of disorder by quantifying the extent of computationally-predicted protein disorder in the core clock of the key eukaryotic circadian model organisms Drosophila melanogaster, Neurospora crassa, and Mus musculus. We further examine previously published work, as well as feature novel experimental evidence, demonstrating that the core negative arm circadian period drivers FREQUENCY (Neurospora crassa) and PERIOD-2 (PER2) (Mus musculus), possess biochemical characteristics of intrinsically disordered proteins. Finally, we discuss the potential contributions of the inherent biophysical principals of intrinsically disordered proteins that may explain the vital mechanistic roles they play in the clock to drive their broad evolutionary conservation in circadian timekeeping. CONCLUSION: The pervasive conservation of disorder amongst the clock in the crown eukaryotes suggests that disorder is essential for optimal circadian timing from fungi to animals, providing vital homeostatic cellular maintenance and coordinating organismal physiology across phylogenetic kingdoms. Video abstract.

Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential.

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.

The circadian system as an organizer of metabolism.

The regulation of metabolism by circadian systems is believed to be a key reason for the extensive representation of circadian rhythms within the tree of life. Despite this, surprisingly little work has focused on the link between metabolism and the clock in Neurospora, a key model system in circadian research. The analysis that has been performed has focused on the unidirectional control from the clock to metabolism and largely ignored the feedback from metabolism on the clock. Recent efforts to understand these links have broken new ground, revealing bidirectional control from the clock to metabolism and vise-versa, showing just how strongly interconnected these two cellular systems can be in fungi. This review describes both well understood and emerging links between the clock and metabolic output of fungi as well as the role that metabolism plays in influencing the rhythm set by the clock.

Circadian regulation of physiology by disordered protein-protein interactions.

Cellular circadian clocks, the molecular timers that coordinate physiology to the day/night cycle across the domains of life, are widely regulated by disordereddisordered protein interactions. Here, we review the disordered-disordered protein interactions in the circadian clock of Neurospora crassa (N. crassa), a filamentous fungus which is a model organism for clocks in higher eukaryotes. We focus on what is known about the interactions between the intrinsically disordered core negative arm protein FREQUENCEY (FRQ), the other proteins comprising the transcription-translation feedback loop, and the proteins that control output. We compare and contrast this model with other models of eukaryotic clocks, illustrating that protein disorder is a conserved and essential mechanism in the maintenance of circadian clock across species.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

NOX2 inhibition enables retention of the circadian clock in BV2 microglia and primary macrophages.

Introduction: Sustained neuroinflammation is a major contributor to the progression of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD) diseases. Neuroinflammation, like other cellular processes, is affected by the circadian clock. Microglia, the resident immune cells in the brain, act as major contributors to neuroinflammation and are under the influence of the circadian clock. Microglial responses such as activation, recruitment, and cytokine expression are rhythmic in their response to various stimuli. While the link between circadian rhythms and neuroinflammation is clear, significant gaps remain in our understanding of this complex relationship. To gain a greater understanding of this relationship, the interaction between the microglial circadian clock and the enzyme NADPH Oxidase Isoform 2 (NOX2) was studied; NOX2 is essential for the production of reactive oxygen species (ROS) in oxidative stress, an integral characteristic of neuroinflammation. Methods: BV2 microglia were examined over circadian time, demonstrating oscillations of the clock genes Per2 and Bmal1 and the NOX2 subunits gp91phox and p47phox. Results: The BV2 microglial clock exerted significant control over NOX2 expression and inhibition of NOX2 enabled the microglia to retain a functional circadian clock while reducing levels of ROS and inflammatory cytokines. These trends were mirrored in mouse bone marrow-derived primary macrophages. Conclusions: NOX2 plays a crucial role in the interaction between the circadian clock and the activation of microglia/macrophages into their pro-inflammatory state, which has important implications in the control of neuroinflammation.

Conformational changes in the negative arm of the circadian clock correlate with dynamic interactomes involved in post-transcriptional regulation.

Biology is tuned to the Earth's diurnal cycle by the circadian clock, a transcriptional/translational negative feedback loop that regulates physiology via transcriptional activation and other post-transcriptional mechanisms. We hypothesize that circadian post-transcriptional regulation might stem from conformational shifts in the intrinsically disordered proteins that comprise the negative arm of the feedback loop to coordinate variation in negative-arm-centered macromolecular complexes. This work demonstrates temporal conformational fluidity in the negative arm that correlates with 24-h variation in physiologically diverse macromolecular complex components in eukaryotic clock proteins. Short linear motifs on the negative-arm proteins that correspond with the interactors localized to disordered regions and known temporal phosphorylation sites suggesting changes in these macromolecular complexes could be due to conformational changes imparted by the temporal phospho-state. Interactors that oscillate in the macromolecular complexes over circadian time correlate with post-transcriptionally regulated proteins, highlighting how time-of-day variation in the negative-arm protein complexes may tune cellular physiology.

Clostridium difficile MazF toxin exhibits selective, not global, mRNA cleavage.

Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.

Bacterial toxin RelE mediates frequent codon-independent mRNA cleavage from the 5' end of coding regions in vivo.

The enzymatic activity of the RelE bacterial toxin component of the Escherichia coli RelBE toxin-antitoxin system has been extensively studied in vitro and to a lesser extent in vivo. These earlier reports revealed that 1) RelE alone does not exhibit mRNA cleavage activity, 2) RelE mediates mRNA cleavage through its association with the ribosome, 3) RelE-mediated mRNA cleavage occurs at the ribosomal A site and, 4) Cleavage of mRNA by RelE exhibits high codon specificity. More specifically, RelE exhibits a preference for the stop codons UAG and UGA and sense codons CAG and UCG in vitro. In this study, we used a comprehensive primer extension approach to map the frequency and codon specificity of RelE cleavage activity in vivo. We found extensive cleavage at the beginning of the coding region of five transcripts, ompA, lpp, ompF, rpsA, and tufA. We then mapped RelE cleavage sites across one short transcript (lpp) and two long transcripts (ompF and ompA). RelE cut all of these transcripts frequently and efficiently within the first ∼100 codons, only occasionally cut beyond this point, and rarely cut at sites in proximity to the 3' end. Among 196 RelE sites in these five transcripts, there was no preference for CAG or UCG sense codons. In fact, bioinformatic analysis of the RelE cleavage sites failed to identify any sequence preferences. These results suggest a model of RelE function distinct from those proposed previously, because RelE directed frequent codon-independent mRNA cleavage coincident with the commencement of translation elongation.

The PAICE suite reveals circadian posttranscriptional timing of noncoding RNAs and spliceosome components in Mus musculus macrophages.

Circadian rhythms broadly regulate physiological functions by tuning oscillations in the levels of mRNAs and proteins to the 24-h day/night cycle. Globally assessing which mRNAs and proteins are timed by the clock necessitates accurate recognition of oscillations in RNA and protein data, particularly in large omics data sets. Tools that employ fixed-amplitude models have previously been used to positive effect. However, the recognition of amplitude change in circadian oscillations required a new generation of analytical software to enhance the identification of these oscillations. To address this gap, we created the Pipeline for Amplitude Integration of Circadian Exploration suite. Here, we demonstrate the Pipeline for Amplitude Integration of Circadian Exploration suite's increased utility to detect circadian trends through the joint modeling of the Mus musculus macrophage transcriptome and proteome. Our enhanced detection confirmed extensive circadian posttranscriptional regulation in macrophages but highlighted that some of the reported discrepancy between mRNA and protein oscillations was due to noise in data. We further applied the Pipeline for Amplitude Integration of Circadian Exploration suite to investigate the circadian timing of noncoding RNAs, documenting extensive circadian timing of long noncoding RNAs and small nuclear RNAs, which control the recognition of mRNA in the spliceosome complex. By tracking oscillating spliceosome complex proteins using the PAICE suite, we noted that the clock broadly regulates the spliceosome, particularly the major spliceosome complex. As most of the above-noted rhythms had damped amplitude changes in their oscillations, this work highlights the importance of the PAICE suite in the thorough enumeration of oscillations in omics-scale datasets.

The circadian clock influences T cell responses to vaccination by regulating dendritic cell antigen processing.

Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca2+ are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.