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.

Neurology podcast utilization during the COVID-19 pandemic.

BACKGROUND: As medical education shifted to a virtual environment during the early coronavirus disease 2019 (COVID-19) pandemic, we evaluated how neurology podcasting may have been utilized during this period, and which features of podcasts have been more highly sought by a medical audience. METHODS: We conducted a retrospective analysis of neurology-themed blogs and/or podcasts between April 2019 and May 2020. Programs were eligible if they reported mean monthly downloads > 2000, were affiliated with an academic society, or offered continuing medical education credit. Thirty-day download counts were compared between study months, with adjustment for multiple testing. Exploratory analyses were performed to determine which podcast features were associated with higher downloads. RESULTS: Of the 12 neurology podcasts surveyed, 8 completed the survey and 5 met inclusion criteria. The median monthly download count was 2865 (IQR 869-7497), with significant variability between programs (p < 0.001). While there was a 358% increase in downloads during April 2020 when compared to the previous month, this was not significant (median 8124 [IQR 2913-14,177] vs. 2268 [IQR 540-6116], padj = 0.80). The non-significant increase in overall downloads during April 2020 corresponded to an increase in unique episodes during that month (r = 0.48, p = 0.003). There was no difference in 30-day downloads among episodes including COVID-19 content versus not (median 1979 [IQR 791-2873] vs. 1171 [IQR 405-2665], p = 0.28). CONCLUSIONS: In this unique, exploratory study of academic neurology-themed podcasts, there was no significant increase in episode downloads during the early COVID-19 pandemic. A more comprehensive analysis of general and subspecialty medical podcasts is underway.

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.

Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions.

Diabetes-induced cardiomyopathy is characterized by cardiac remodeling, fibrosis, and endothelial dysfunction, with no treatment options currently available. Hyperglycemic memory by endothelial cells may play the key role in microvascular complications in diabetes, providing a potential target for therapeutic approaches. This study tested the hypothesis that a proangiogenic environment can augment diabetes-induced deficiencies in endothelial cell angiogenic and biomechanical responses. Endothelial responses were quantified for two models of diabetic conditions: 1) an in vitro acute and chronic hyperglycemia where normal cardiac endothelial cells were exposed to high-glucose media, and 2) an in vivo chronic diabetes model where the cells were isolated from rats with type I streptozotocin-induced diabetes. Capillary morphogenesis, VEGF and nitric oxide expression, cell morphology, orientation, proliferation, and apoptosis were determined for cells cultured on Matrigel or proangiogenic nanofiber hydrogel. The effects of biomechanical stimulation were assessed following cell exposure to uniaxial strain. The results demonstrate that diabetes alters cardiac endothelium angiogenic response, with differential effects of acute and chronic exposure to high-glucose conditions, consistent with the concept that endothelial cells may have a long-term "hyperglycemic memory" of the physiological environment in the body. Furthermore, endothelial cell exposure to strain significantly diminishes their angiogenic potential following strain application. Both diabetes and strain-associated deficiencies can be augmented in the proangiogenic nanofiber microenvironment. These findings may contribute to the development of novel approaches to reverse hyperglycemic memory of endothelium and enhance vascularization of the diabetic heart, where improved angiogenic and biomechanical responses can be the key factor to successful therapy.

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.