James W. Checco

Large-scale radiation catastrophes and localized exposure incidents remain major threats to public health. Effective treatment following radiation exposure is challenging because radiation dose, timing, and affected tissues can vary widely between individuals. To better understand the biological response to radiation injury, we employed a multi-omics approach integrating transcriptomics, proteomics, and metabolomics. In this study, mouse bone marrow was analyzed at 1, 6, and 24 h after whole-body irradiation and compared with bone marrow from non-irradiated mice. Multivariate and univariate analyses revealed time-dependent changes across transcriptomic, proteomic, and metabolomic profiles. We further applied machine learning-based integrative analysis and biomarker discovery approaches to identify key molecular responses associated with radiation exposure. Integrated analysis showed that the acute radiation response in bone marrow included immune-related processes, followed by intermediate metabolic reprogramming, and later disruptions in cell cycle regulation. Biomarker analysis identified several promising candidates, including the transcripts Sapcd2 and Cd28, proteins FRIL1 and CDK1, and the metabolites 3-hydroxyisobutyrate and taurine. Overall, our findings demonstrate that bone marrow from irradiated mice undergoes dynamic and coordinated molecular changes over time. These responses include activation of ferroptosis defense mechanisms, mitotic dysfunction, and pathways associated with programmed cell death.

Proliferating cell nuclear antigen (PCNA) is an essential sliding clamp that coordinates nearly all DNA-templated processes. It does so by recruiting a diverse array of factors to DNA through PCNA-interacting protein (PIP) motifs on PCNA-binding proteins. Chromatin assembly factor 1 (CAF-1) is a histone chaperone that deposits histones onto silent regions of the genome immediately following DNA replication. PCNA recruitment of CAF-1 to the replication fork is essential for nucleosome assembly and epigenetic inheritance. In yeast, CAF-1 recruitment to PCNA is assumed to be facilitated using one PIP motif. Here, we identify a second PIP motif in CAF-1, designated PIP1, located within the N-terminal intrinsically disordered region of the protein. Binding kinetics demonstrate that the PIP1 motif sequence binds PCNA with substantially lower affinity than the previously characterized PIP motif, designated PIP2. Structural and binding data reveal PIP1 as an extended PIP motif, in which C-terminal flanking residues make extensive, atypical contacts with PCNA that significantly increase its affinity. Neither PIP1 nor PIP2 alone is required for CAF-1–mediated gene silencing in vivo, but simultaneous disruption of both abolishes CAF-1 function. Together, these findings suggest CAF-1 engages PCNA multivalently and provide new insight into how PCNA selectively recognizes binding partners during nucleosome assembly.

Proliferating cell nuclear antigen (PCNA) is an essential sliding clamp that coordinates nearly all DNA-templated processes. It does so by recruiting a diverse array of factors to DNA through PCNA-interacting protein (PIP) motifs on PCNA-binding proteins. Chromatin assembly factor 1 (CAF-1) is a histone chaperone that deposits histones onto silent regions of the genome immediately following DNA replication. PCNA recruitment of CAF-1 to the replication fork is essential for nucleosome assembly and epigenetic inheritance. In yeast, CAF-1 recruitment to PCNA is assumed to be facilitated using one PIP motif. Here, we identify a second PIP motif in CAF-1, designated PIP1, located within the N-terminal intrinsically disordered region of the protein. Binding kinetics demon-strate the PIP1 motif sequence binds PCNA with substantially lower affinity than the previously characterized PIP motif, designated PIP2. Structural and binding data reveal PIP1 as an extended PIP motif, where downstream residues make extensive, atypical contacts with PCNA that significantly increase its affinity. Neither PIP1 nor PIP2 alone is required for CAF-1–mediated gene silencing in vivo, but simultaneous disruption of both abolishes CAF-1 function. Together, these findings suggest CAF-1 engages PCNA multivalently and provide new insight into how PCNA selectively recognizes binding partners during nucleosome assembly.

Acute radiation syndrome (ARS) occurs when hematopoietic or gastrointestinal cells are damaged by radiation exposure causing DNA damage to the bone marrow and gastrointestinal epithelial stem cell populations. In these highly proliferative cell types, DNA damage inhibits stem cell repopulation. In humans and animals, this inability to regenerate stem cells is lethal. Within this manuscript, several compounds, Amifostine, Captopril, Ciprofloxacin, PrC-210, 5-AED (5-androstene-3β,17β-diol), and 5-AET (5-androstene-3β,7β,17B-triol), are assessed for their ability to protect against ARS in an in vitro and/or in vivo setting. ARS was accomplished by irradiating mouse bone marrow cells or rat intestinal epithelial (IEC-6) cells in vitro with 4–8 Gy and in vivo by exposing Mus musculus to 7.3 Gy of whole-body irradiation. The primary endpoints of this study include cellular viability, DNA damage via γ-H2AX, colony formation, and overall survival at 30-days post-irradiation. In addition to evaluating the radioprotective performance of each compound, this study establishes a distinct set of in vitro assays to predict the overall efficacy of potential radioprotectors in an in vivo model of ARS. Furthermore, these results highlight the need for FDA-approved medical intervention to protect against ARS.

Hibernating mammals such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experience significant reductions in oxidative metabolism and body temperature when entering a state known as torpor. Animals entering or exiting torpor do not experience permanent loss of brain function or other injuries, and the processes that enable such neuroprotection are not well understood. To gain insight into changes in protein function that occur in the dramatically different physiological states of hibernation, we performed quantitative phosphoproteomics experiments on thirteen-lined ground squirrels that are summer-active, winter-torpid, and spring-active. An important aspect of our approach was the use of focused microwave irradiation of the brain to sacrifice the animals and rapidly inactivate phosphatases and kinases to preserve the native phosphoproteome. Overall, our results showed pronounced changes in phosphorylated proteins for the transitions into and out of torpor, including proteins involved in gene expression, DNA maintenance and repair, cellular plasticity, and human disease. In contrast, the transition between the active states showed minimal changes. This study offers valuable insight into the global changes in brain phosphorylation in hibernating mammals, the results of which may be relevant to future therapeutic strategies for brain injury.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

Liquid chromatography–mass spectrometry (LC-MS)-based peptidomics methods allow for the detection and identification of many peptides in a complex biological mixture in an untargeted manner. Quantitative peptidomics approaches allow for comparisons of peptide abundance between different samples, allowing one to draw conclusions about peptide differences as a function of experimental treatment or physiology. While stable isotope labeling is a powerful approach for quantitative proteomics and peptidomics, advances in mass spectrometry instrumentation and analysis tools have allowed label-free methods to gain popularity in recent years. In a general label-free quantitative peptidomics experiment, peak intensity information for each peptide is compared across multiple LC-MS runs. Here, we outline a general approach for label-free quantitative peptidomics experiments, including steps for sample preparation, LC-MS data acquisition, data processing, and statistical analysis. Special attention is paid to address run-to-run variability, which can lead to several major problems in label-free experiments. Overall, our method provides researchers with a framework for the development of their own quantitative peptidomics workflows applicable to quantitation of peptides from a wide variety of different biological sources. Key words Peptidomics Label-free Peptides Mass spectrometry LC-MS Run-to-run variability Missing values Pooled QC samples

Hibernation in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) takes place over 4-6 months and is characterized by multiday bouts of hypothermic torpor (5-7 °C core body temperature) that are regularly interrupted every 1-2 weeks by brief (12-24 h) normothermic active periods called interbout arousals. Our goal was to gain insight into the molecular mechanisms that underlie the hibernator's ability to preserve heart function and avoid the deleterious effects of skeletal muscle disuse atrophy over prolonged periods of inactivity, starvation, and near-freezing body temperatures. To achieve this goal, we performed organelle enrichment of heart and skeletal muscle at five seasonal time points followed by LC-MS-based label-free quantitative proteomics. In both organs, we saw an increase in the levels of many proteins as ground squirrels transition from an active state to a prehibernation state in the fall. Interestingly, seasonal abundance patterns identified DHRS7C, SRL, TRIM72, RTN2, and MPZ as potential protein candidates for mitigating disuse atrophy in skeletal muscle, and ex vivo contractile mechanics analysis revealed no deleterious effects in the ground squirrel's muscles despite prolongedthirte sedentary activity. Overall, an increased understanding of protein abundance in hibernators may enable novel therapeutic strategies to treat muscle disuse atrophy and heart disease in humans.

Prohormone-derived neuropeptides act as cell-cell signaling molecules to mediate a wide variety of biological processes in the animal brain. Mass spectrometry-based peptidomic experiments are valuable approaches to gain insight into the dynamics of individual peptides under different physiological conditions or experimental treatments. However, the use of anesthetics during animal procedures may confound experimental peptide measurements, especially in the brain, where anesthetics act. Here, we investigated the effects of the commonly used anesthetics isoflurane and sodium pentobarbital on the peptide profile in the rodent hypothalamus and cerebral cortex, as assessed by label-free quantitative peptidomics. Our results showed that neither anesthetic dramatically alters peptide levels, although extended isoflurane exposure did cause changes in a small number of prohormone-derived peptides in the cerebral cortex. Overall, our results demonstrate that acute anesthetic administration can be utilized in peptidomic experiments of the hypothalamus and cerebral cortex without greatly affecting the measured peptide profiles.