bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatiotemporal variation of ringed seal blubber cortisol levels in the Canadian Arctic
Authors: Ogloff Wesley R , Anderson Randi A , Yurkowski David J , Debets Cassandra D , Anderson W Gary , Ferguson Steven H
Published: May 2022

Climate change in the Arctic has widespread and complex effects on the health of animals and their populations. We used radioimmunoassay to measure blubber cortisol in ringed seals (Pusa hispida) sampled in Ulukhaktok, Inuvialuit Settlement Region, Northwest Territories, Canada (spring, 2002, 2004–2005, 2007–2012) and Arviat, Nunavut, Canada (autumn, 2003–2012) to examine chronic stress relative to biology (age, sex, length), body condition (blubber depth), and diet (δ13C, δ15N, and isotopic niche size). Ulukhaktok ringed seals had higher cortisol concentrations overall (0.46 ± 0.04 ng/g) than Arviat ringed seals (0.36 ± 0.03 ng/g), and these higher concentrations were associated with higher muscle δ15N and lower blubber thickness. In contrast, blubber cortisol concentrations for Arviat ringed seals decreased with blubber depth and increased with age, though testing of age effects individually suggests that age-related patterns are weak. Annual mean cortisol concentration increased from 2003 to 2012 in Arviat ringed seals, but low sample sizes precluded analysis of annual patterns for Ulukhaktok ringed seals. The trend of increased cortisol over time in Arviat ringed seals suggests that they might be experiencing greater chronic stress over time, which could have implications for numerous population health metrics including reproduction and pup recruitment.
Design and fabrication of an electrochemical chip for liquid-phase transmission electron microscopy
Authors: Sasaki Yuki , Mizushima Ayako , Mita Yoshio , Yoshida Kaname , Kuwabara Akihide , Ikuhara Yuichi
Published: May 2022

Liquid-phase transmission electron microscopy (LP-TEM) can be used with an electrochemical chip (e-chip) to observe electrochemical reactions in a liquid in situ. The design of electrodes on an e-chip fabricated using microelectromechanical system technology cannot be easily changed. Here, we report a newly designed e-chip and its fabrication process. Electrodes with a desired shape were fabricated with various metals via an additional step of vacuum deposition onto our e-chip with a shadow mask. For precise control of the electrochemical reactions in LP-TEM, optimization of the electrode shape and material is critical.
Evolution, Expression Patterns, and Distribution of Novel Ribbon Worm Predatory and Defensive Toxins
Authors: Verdes Aida , Taboada Sergi , Hamilton Brett R. , Undheim Eivind A.B. , Sonoda Gabriel G. , Andrade Sonia C.S. , Morato Esperanza , Marina Ana Isabel , Cárdenas César A. , Riesgo Ana
Published: May 2022

Ribbon worms are active predators that use an eversible proboscis to inject venom into their prey and defend themselves with toxic epidermal secretions. Previous work on nemertean venom has largely focused on just a few species and has not investigated the different predatory and defensive secretions in detail. Consequently, our understanding of the composition and evolution of ribbon worm venoms is still very limited. Here, we present a comparative study of nemertean venom combining RNA-seq differential gene expression analyses of venom-producing tissues, tandem mass spectrometry-based proteomics of toxic secretions, and mass spectrometry imaging of proboscis sections, to shed light onto the composition and evolution of predatory and defensive toxic secretions in Antarctonemertes valida. Our analyses reveal a wide diversity of putative defensive and predatory toxins with tissue-specific gene expression patterns and restricted distributions to the mucus and proboscis proteomes respectively, suggesting that ribbon worms produce distinct toxin cocktails for predation and defense. Our results also highlight the presence of numerous lineage-specific toxins, indicating that venom evolution is highly divergent across nemerteans, producing toxin cocktails that might be finely tuned to subdue different prey. Our data also suggest that the hoplonemertean proboscis is a highly specialized predatory organ that seems to be involved in a variety of biological functions besides predation, including secretion and sensory perception. Overall, our results advance our knowledge into the diversity and evolution of nemertean venoms and highlight the importance of combining different types of data to characterize toxin composition in understudied venomous organisms.
The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola
Authors: Liu Wenkui , Han Lu , Chen Jinzhu , Liang Xiaofei , Wang Bo , Gleason Mark L. , Zhang Rong , Sun Guangyu
Published: May 2022

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant Δ CfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, Δ CfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. Δ CfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of Δ CfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development ( CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation ( CfCBP1 and CfCHS7), pectin degradation ( CfPelA and CfPelB), sexual development ( CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis ( CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.
Dietary Diversity and Evolution of Cranial Adaptations in Turtles
Authors: Smith Heather F.
Published: May 2022

The evolution of the turtle skull has involved a series of unique modifications resulting in increased bite force stemming from cranial elongation, rearrangement of jaw adductor musculature, and reduction of cranial kinesis. Loss of teeth, development of the keratinous rhamphotheca, and evolution of a highly flexible neck have also driven turtle cranial evolution. Dietary diversity across the clade has resulted in a diverse range of morphological variations of the turtle cranial Bauplan, including those associated with suction feeding and tongue‐dependent food uptake.

The Cenomanian Arlington Archosaur Site (AAS) from the Woodbine Formation of Texas was home to numerous mid‐Cretaceous turtle taxa. This diverse faunal assemblage allows an evaluation of how dietary adaptations can facilitate partitioning of the available niche space among co‐occurring turtle species. Baenids, such as Trinitichelys maini at AAS, are typically reconstructed as being primarily carnivorous, although there is evidence that some taxa were molluscivorous. The AAS helochelydrid Naomichelys was likely a generalized omnivore, being more terrestrial than the other AAS turtle taxa. Bothremydids, such as the AAS species Pleurochayah appalachius, have a wide range of dietary specializations including durophagy and suction‐feeding. These adaptations are evidenced by the broad range of bothremydid cranial morphologies including expanded triturating surfaces and an inflated face. Highly aquatic trionychids are predominantly piscivorous, although they will also opportunistically hunt other small prey such crustaceans and amphibians. Many trionychid species must be fully submerged under water in order to properly swallow their food in contrast to some semi‐aquatic turtles. A newly identified protostegid turtle reveals a marine component to the AAS turtle fauna, suggesting the presence of an open ocean piscivore. Finally, an intriguing new lower jaw with well‐developed denticles indicates the presence of an obligate herbivore among the AAS turtle fauna. This diverse array of dietary proclivities and adaptations reveals how AAS turtle communities divided the available ecospace in order to avoid interspecific competition for available resources among turtle taxa and with the other AAS herpetofauna.
Sex differences in lung inflammation and microbiome profiles in asthmatic mice exposed to ambient ozone
Authors: Colon Montañez Keishla N. , Fuentes Nathalie , Montes Deborah , Silveyra Patricia
Published: May 2022

Ambient ozone, a product of photochemical reactions between volatile organic compounds and nitrogen oxides, is known to be one of the most dangerous air pollutants. Ozone inhalation can aggravate preexisting inflammatory lung diseases such as asthma, which are more frequently diagnosed in females than males. Despite this, the molecular mechanisms underlying the effects of ozone in the male and female lung have yet to be discovered. We hypothesized that exposure to ozone exerts differential inflammatory and immune responses in the lungs of male and females with asthma. To examine this, we treated adult male and female C57BL/6J mice with an allergen (house dust mite extract) intranasally for 5 weeks to trigger asthma. We then exposed mice to 2 ppm of ozone or filtered air (FA) for 3 hours, and collected lung tissue 24 hours later. We extracted lung RNA, and measure the expression of 92 immune response associated genes by PCR with the TaqMan® Array 96‐well Mouse Immune Response Plate (ThermoFisher). We also extracted DNA to determined lung microbiome profiles via 16S rRNA amplicon sequencing using a MiSeq instrument. The resulting paired‐end reads were processed using QIIME 2 2018.11. Amplicon sequencing units from DADA2 were assigned taxonomic identifiers based on the GreenGenes database release 13_08 using the QIIME 2 q2‐featureclassifier. Results were summarized, visualized through Principal Coordinate Analysis, and significance was estimated as implemented in QIIME2. Our preliminary results show that, after exposure to ozone, asthmatic male mice had higher lung expression of immune response genes (cell surface receptors and signaling molecules) whereas asthmatic females had higher expression of pro‐inflammatory cytokines, transcription factors, and regulators of immunity than mice exposed to FA. We also identified differential microbiome profiles between male and female asthmatic mice and in response to ozone, including a higher content of Firmicutes and a higher Firmicutes/Bacteriodetes ratio in males than females. Moreover, asthmatic females displayed a more pronounced lung bacterial diversity and higher relative abundance of Proteobacteria when compared to males. We conclude that ozone exposure triggers differential inflammatory/immune mechanisms in the male and female lungs of asthmatic mice, and that these mechanisms could be mediated by lung resident microbes.
Comparison of the Safety and Efficacy of Tofacitinib and Fingolimod in TNBS‐Induced Colitis Model in Adult Zebrafish: The Role of Myd88/NF‐κB/TNF‐α Signaling Pathway
Authors: Mousavi Taraneh , Hassani Shokoufeh , Gholami Mahdi , Vakhshiteh Faezeh , Baeeri Maryam , Rahimifard Mahban , Ghafour‐Broujerdi Elmira , Abdollahi Mohammad
Published: May 2022

Due to the inadequate response, side effects, parenteral route of administration, and immunogenicity, oral targeted small molecules seem to replace monoclonal antibodies in the future management of ulcerative colitis (UC). In order to pave the way for clinical translations of small molecules in UC, for the first time, we compared the efficacy of fingolimod and tofacitinib in preclinical model of colitis, and uncovered another mechanism of action that these medicines may act through.

We implicated adult zebrafish to find out its pros/cons, investigated aquatic toxicity of the mentioned compounds, and designed a procedure to evaluate compound's efficacy in adult zebrafish colitis model; hence, facilitating screening of further agents.

Tofacitinib and fingolimod were investigated to be in category 1 of Globally Harmonized System (GHS) aquatic toxicity classification with the LC50 of 0.9014 and 0.36 mg/L, respectively, based on the acute toxicity assessment within 96 h.

Considering efficacy assessments, colitis was induced through intrarectal administration of 160 mM TNBS for 48 h. Then, animals were exposed to different concentrations of tofacitinib (0.25, 0.57, 0.72, 0.90 mg/L) and fingolimod (0.23, 0.28, 0.36, 0.45 mg/L) for 96 h. Subsequent analyses revealed that TNBS significantly reduced length of villi, number of goblet cells, level of TNF‐α, MyD88, and NF‐κB2, increased thickness of villi and necrosis, and induced histopathological changes, including intestinal edema, submucosal hemorrhage/hyperemia with the presence of inflammatory cells. All the aforementioned parameters were considerably enhanced almost dose‐dependently with both agents, wherein the effect of 0.45 mg/L fingolimod was superior to other groups. As well, there were no signs of hyperemia/hemorrhage in harvested intestinal samples. Resultantly, we revealed that these medications may suppress canonical and non‐canonical NF‐κB pathways through targeting toll‐like receptors and MyD88.

Given the better efficacy and multi‐targeted action of fingolimod, it worth to focus on sphingosine‐1 phosphate receptor modulators in clinical trials of UC and compare them with other classes of medications; albeit, their ecotoxicity aspect should not be overlooked.
Investigating the Metabolism of Melanocyte Development
Authors: Asencio Torres Gabriela , Johns Eleanor , White Richard
Published: May 2022

During development, melanocytes undergo a series of cellular and metabolic changes in order to reach a terminally differentiated state. Some of these changes include extensive proliferation, formation of dendritic projections from the cell membrane, and melanin synthesis. Each of these processes is likely to create distinct metabolic needs, however, little work has been done to investigate how the differentiating melanocyte meets these requirements. Previous work in our lab identified that stromal mesenchymal adipocytes are donors of lipids that aids in melanoma progression and metastasis. This raises the question of whether fatty acid metabolism might also play a role in melanocyte development. Additionally, it indicates a need for further investigation into the role of cellular metabolism in both melanocyte differentiation and related diseases. Herein, we performed a chemical screen as a discovery approach using the Sigma Aldrich LOPAC library containing 1280 compounds in T5D zebrafish embryos and validate the primary hits via titration at different developmental time points. Treatment was initiated at 24‐hour post fertilization for a 48‐hour period at a 10 μM concentration. With this screen we identified a total of fifty compounds which affected melanocyte growth, development, migration pattern, and/or phenotypic appearance in the context of a complete developing organism allowing a more comprehensive analysis. Preliminary data suggest that different pathways such as T‐Cell Receptor inhibition, nitric oxide donors and/or sequesters, inhibition of inflammatory responses from macrophages, basophils and mast cells can potentially affect melanocyte development. Together, these findings help us to better understand the details of melanocyte development and further characterization of these pathways will greatly enhance our understanding of the metabolic requirements of these processes and could yield insight into the role of these processes in melanoma and other disease contexts.
PP2A and LRP‐1 targeted protection of human retinal pigment epithelial cells
Authors: Starghill Micah T. , Batiste Malia , Bowden Samantha , Davenport Ian , Bhattacharjee Partha
Published: May 2022

Retinal pigment epithelial (RPE) cell disorders contribute to the leading causes of vision loss in the United States. Diabetic retinopathy and Age‐related macular degeneration are RPE disorders that develop as a consequence to the accumulation of toxic retinal lipids. However, the mechanism of how RPE regulates lipid homeostasis in response to intra‐retinal dysfunction of lipid metabolism is unknown. The purpose of this project is to provide new proof of principle information, toward the understanding, of how RPE regulates oxidative stress induced alteration of intracellular phosphatase/kinase signaling leading to retinal disorders. Through confocal immunofluorescence imaging, transepithelial resistance (TEER) measurement and Western Blot analysis of protein expression of primary human RPE(hRPE) cells are investigated after the treatment of lipid peroxidation products. Protein phosphatase 2A (PP2A), appears to have a pertinent role in the regulation of kinases (p‐Akt and p‐ERK ½) associated to RPE loss and TEER maintenance in response to oxidative stress induced by lipid peroxidation products. Altered PP2A signaling was found to be modulated by low density lipoprotein related receptor protein‐1 (LRP‐1) targeted peptidomimetic treatment. This project also provides potential clinical application to the treatment of retinal degeneration associated with intraretinal lipid homeostasis using a patented peptidomimetic targeting LRP‐1 and PP2A signaling.
Mirtazapine seeking and withdrawal in free‐choice drinking model
Authors: Althobaiti Yusuf S.
Published: May 2022

Drug addiction is one of the major public health concerns around the world. Addiction to prescription medications has been growing due to the psychoactive effects of these medications, availabilities, low price and no legal consequences were practiced against the abusers. One of these prescription medications is mirtazapine (MIRT). MIRT is an antidepressant that is recently reported in different case studies to be abused and could induce withdrawal symptoms. No previous study has investigated its abuse potential in animal model of drug addiction. Here we conducted a free‐choice drinking paradigm to investigate the voluntary drinking of MIRT in two different concentrations. Male bulb/c mice were allowed free access to two bottles of water for five days and their intake and body weight were recorded daily to calculate, based on consumed fluid, the concentrations of MIRT solutions. Mice were then divided into three groups; the first group had free access to two bottles of water. The second group (MIRT‐10) had free access to one bottle of water and one bottle of MIRT in a concentration that might yield daily intake of (10 mg/kg). The third group (MIRT‐20) had free access to one bottle of water and one bottle of MIRT in a concentration that might yield daily intake of (20 mg/kg). Interestingly, animals in MIRT‐20 showed a significant increase in MIRT intake as compared to MIRT‐10 group indicating that MIRT in higher doses can induce drug seeking effects. Following the drinking phase, and to confirm any effects on memory and recognition, animals underwent Novel Object Recognition test (NORT). Animals in MIRT‐20 showed a significant deterioration in the recognition of the novel objects as compared to the control and MIRT‐10 groups. This indicates the destructive nature of abusing MIRT on recognition and memory which is consistent with the effects of different drugs of abuse. All animals were then only exposed to two bottles of water to induce withdrawal symptoms. After five days of abstinence, animals in MIRT‐20 group showed a significant increase in the immobility time in forced swimming test and tail suspension test as compared to the control and MIRT‐10. This indicates that animals in MIRT‐20 might have depressive‐like symptoms as one of the indicators of withdrawal symptoms related to drug addiction. Together, MIRT was able to induce drug seeking behavior, deteriorate recognition, and cause withdrawal symptoms. This might confirm that MIRT has potential for inducing drug dependence and its use should be controlled by legal authorities. Further studies are needed to investigate the neurobiological basis of MIRT‐induced drug seeking behavior.
The effect of photoperiod on the serotoninergic system of the thymus and its role in implementing the effects of exogenous melatonin
Authors: Luzikova Elena M. , Sergeeva Valentina E. , Sergeev Pavel V. , Lukachev Ivan A.
Published: May 2022

Serotonin‐containing cells of the thymus in Wistar rats were studied, which were kept during 4 weeks of the experiment under the conditions of a natural photoperiod, under the conditions of constant blackout, under the conditions of continuous illumination. The effect of melatonin (“Melaxen” Unipharm, Inc., USA) ad libitum at a concentration of 4 mg / liter with drinking for four weeks) on serotonin‐positive cells of the thymus was studied.

Results

The absence of light does not affect serotonin synthesis in the amine‐producing cells of the diffuse endocrine system in the border of the cortical and medullary lobules, but it suppresses the amine‐absorbing properties in the cortical substance cells of the thymus lobules. Continuous illumination does not affect the amine‐absorbing properties of the cells of the diffuse endocrine system in the cortical lobular substance, but promotes serotonin accumulation in the amine‐producing cells at the border of the thymus cortical and medullary lobules. Regardless of the photoperiod, exogenous melatonin increases the content of serotonin in the cells of the thymic diffuse endocrine system, which is more pronounced in continuous light conditions.

Conclusions

Our studies have shown that changes in the photoperiod stimulate the serotoninergic system of the thymus, and exogenous melatonin smooths out these effects.
Down‐expression of MiR30a‐5p promotes proliferation and migration of Non‐Small Cell Lung Cancer cells by targetting MTDH
Authors: Tapara Dramani Maman Souraka
Published: May 2022

OBJECTIVE

MTDH has been identified to play a critical role in many cancer types, but its potential mechanism in Non‐Small Cell Lung Cancer (NSCLC) still remains unclear. We investigated the role of miRNA30a‐5p in the regulation of MTDH expression, which is known as an essential biomarker for NSCLC initiation and progression.

MATERIALS AND METHODS

Quantitative real‐time PCR (qRT‐PCR) has been performed to evaluate the expression level of MTDH and miR30a‐5p. Then, we transfected miR30a‐5p mimic and inhibitor into H1299 and A549 cells to investigate their impact in cell proliferation, migration, and invasion by using CCK‐8 assay, wound‐healing assay, and transwell assay respectively. The evidence of miR30a‐5p as a specific target of the MTDH gene was explored through luciferase report assay. The expression level of MTDH was evaluated by its mRNA and protein level using a western blot assay.

RESULTS

Results revealed that miR30a‐5p was upregulated whereas MTDH was downregulated. Down‐expression of miR30a‐5p increases the expression level of MTDH and promotes NSCLC cell proliferation and invasion. Luciferase report assay confirmed the binding of miR30a‐5p on MTDH mRNA 3′UTR region.

CONCLUSIONS

This funding highlights the silencing mechanism of MTDH in lung cancer and further revealed that MTDH can be used as a potential diagnostic and therapeutic biomarker.
Structure‐activity relationships for several series of fragment‐based inhibitors that target Trichomonas vaginalis nucleoside ribohydrolase enzymes
Authors: Saljanin Edina , Ajmal Erum , Vanegas Davi , VanAlstine‐Parris Melissa , Stockman Brian
Published: May 2022

Trichomoniasis, the most common sexually transmitted infection in the world, is caused by the parasitic protozoan Trichomonas vaginalis. Upon infection, T. vaginalis has been known to cause a variety of adverse effects as well as predisposition to other illnesses. Trichomoniasis is commonly treated with 5‐nitroimidazole drugs; however, some strains have developed resistance to these drugs and novel treatments are needed. Since T. vaginalis cannot synthesize nucleobases de novo, it relies on nucleoside ribohydrolases within its pyrimidine and purine salvage pathways in order to acquire nucleobases from its host. Nucleoside ribohydrolases, such as adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH), cleave the N‐glycosidic bond of purine and pyrimidine nucleosides resulting in the formation of a nucleobase and a ribose sugar. Inhibiting the AGNH or UNH enzymes would block the salvage pathway resulting in parasite cell death. Fragment screening of AGNH and UNH identified ligand‐efficient scaffolds to serve as medicinal chemistry starting points for drug design. A variety of phenyl pyridine, phenyl pyrimidine, and phenyl pyrazine compounds were then synthesized and screened for inhibitory properties. 1H and 19F NMR‐based activity assays were employed, using adenosine and 5‐fluorouridine as substrates for AGNH and UNH, respectively. It was found that compounds containing a hydroxyl group at the 2‐position resulted in lower IC50 values for AGNH, and that the UNH IC50 was lower for compounds with hydroxy or methoxy functional groups at the 2’ and 3’ positions. With the enzyme inhibition data at hand structure‐activity relationships are being derived and will be used in combination with molecular modeling to design the next generation of compounds to be synthesized. Structural data is also being pursued for both enzymes to guide compound design. Highly purified AGNH and UNH, including selenomethionine‐AGNH, have been prepared for crystallography collaborations.
Structural investigations of arginyltransferases
Authors: Van Verna
Published: May 2022

Arginyltransferases (ATE1s) are a class of essential eukaryotic enzymes that catalyze arginylation, the post‐translational transfer of Arg from an aminoacylated tRNA to a range of protein targets. This modification typically occurs at N‐terminal acidic residues, though ATE1 can also covalently attach Arg to mid‐chain residues. Arginylation may have either degradative or non‐degradative effects. For example, some arginylated proteins are processed through the Arg N‐degron pathway, which marks these post‐translationally modified proteins for degradation by the ubiquitin‐proteasome system. In contrast, arginylation may also manifest non‐degradative effects such as thermodynamic stability, subcellular relegalization, or functional changes. The diversity of ATE1’s targets confers its role as a global regulator, influencing functions such as cardiovascular development, neurological processing, and even the stress response. However, a lack of ATE1 structural knowledge has limited the determination of its three‐dimensional fold, how it is regulated, and how it recognizes its substrates. Using a combination of X‐ray crystallography, cryo‐EM, and size‐exclusion chromatography‐coupled small angle X‐ray scattering (SEC‐SAXS), our lab has successfully solved the structure of Saccharomyces cerevisiae ATE1 (ScATE1). The three‐dimensional structure of ScATE1 reveals a bilobed protein containing a canonical GCN5‐related N‐acetyltransferase (GNAT) fold. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding. Furthermore, the structure reveals the spatial connectivity of the N‐terminal domain, which we previously showed binds an [Fe‐S] cluster, to the enzymatic active site, hinting at the cluster’s regulatory influence. As the first atomic‐level structure of any ATE1, this achievement brings us closer to answering pressing questions regarding the molecular‐level mechanism of eukaryotic post‐translational arginylation.
Structure and Dynamics of Human Perilipin 3 Membrane Association
Authors: Choi Yong‐Mi , Fleming Kaelin , Burke John , Airola Michael V.
Published: May 2022

Lipid droplets (LDs) are a unique organelle that contain an oil core mainly composed of triglycerides that is surrounded by a phospholipid monolayer and coated by LD‐associated proteins called perilipins (PLINs). There are five PLINs in human that generally contain a highly conserved N‐terminal PAT domain of unclear function, followed by variable stretches of amphipathic helices previously implicated in LD association, and a C‐terminal 4‐helix bundle domain. During the process of LD biogenesis, perilipin 3 (PLIN3) is the first PLIN to be recruited to nascent LDs as they emerge from the endoplasmic reticulum. However, how PLIN3 recruitment to LDs is affected by lipid composition, and the structure and dynamics of PLIN membrane association has not been examined in detail. Here, using liposome co‐sedimentation and LD‐flotation assays we found that PLIN3 membrane association was enhanced by membrane packing defects and further promoted by the triglyceride precursors diacylglycerol (DAG) and phosphatidic acid. Hydrogen‐deuterium exchange mass spectrometry experiments identified that both the PAT domain and amphipathic helices undergo a transition upon membrane binding from intrinsically disordered to an ordered state. Using constructs containing different portions of the PAT domain and amphipathic helices, we identify a new functional PAT domain that contains all conserved residues and is strongly recruited to DAG enriched membranes. Consistent with these findings, this newly determined functional PAT domain is predicted to form a triangular helical tertiary structure by alphafold and RoseTTAfold. Taken together, this study reveals molecular details of PLIN3 recruitment to membranes and suggests a role for the PAT domain as a major driver of PLIN3 to DAG enriched membranes, which is consistent with the localization of PLIN3 on early nascent LDs.
Pyridoxal phosphate‐dependent reactions in natural products biosynthesis
Authors: Ryan Katherine
Published: May 2022

Pyridoxal 5′‐phosphate (PLP)‐dependent enzymes are widespread in nature. Most reactions catalyzed by PLP‐dependent enzymes traverse through a carbanionic intermediate, which is stabilized by the PLP cofactor. Like all enzymes that deal with carbanions, PLP‐dependent enzymes can be subject to reaction with electrophiles like O2. However, the majority reactions of PLP‐dependent enzymes with O2 are paracatalytic.

Recent studies have identified PLP‐dependent enzymes that employ O2 as a co‐substrate for oxidation of arginine. Such enzymes are part of biosynthetic pathways that convert arginine into heterocycle‐containing natural products. In this talk, I will discuss the discovery of these arginine oxidases, and I will describe what we have learned about their mechanisms. I will furthermore highlight how we characterized an O2‐, PLP‐dependent enzyme encoded by an unknown gene cluster and then used this discovery as a starting point to reconstitute the biosynthetic pathway to the antibiotic azomycin.
Proteome‐scale amino‐acid resolution footprinting of protein‐binding sites in the intrinsically disordered regions
Authors: Ivarsson Ylva
Published: May 2022

Protein‐protein interactions are central to cell function. Numerous studies have provided large‐scale information on human protein‐protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type‐specific interactions disproportionately under‐represented. I will present our efforts towards finding the short linear motif (SLiM)‐based interactions of a variety of protein domains. I will present an optimized proteomic peptide‐phage display (ProP‐PD) library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay, and the tools and guidelines we have defined for processing the data (1). Using the approach we identified >2,000 interaction pairs for 35 known SLiM‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. The amino acid resolution binding site information can be used to pin‐point functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. Finally, I will describe how we developed the approach for large‐scale discovery of coronavirus‐host factor protein interactions (2). We screened more than 130 protein domains for binding to viral peptides, and translated the high‐resolution information on direct virus‐host interactions to a specific peptide‐based antiviral inhibitor of an interaction between the G3BP1/2 proteins and an ΦxFG peptide motif in the SARS‐CoV‐2 nucleocapsid (N) protein. ProP‐PD may thus be used both to illuminate the motif‐based part of the interactome and to uncover leads for innovative inhibitor design.

References

Benz et al., (2022) Proteome‐scale mapping of binding sites in the unstructured regions of the human proteome. Mol Syst Biol. 2022 Jan;18(1):e10584.

Kruse et al., (2021) Large scale discovery of coronavirus‐host factor protein interaction motifs reveals SARS‐CoV‐2 specific mechanisms and vulnerabilities. Nat Commun. 2021 Nov 19;12(1):6761.
Repairing enzymes using spare parts
Authors: Drennan Catherine L. , Andorfer Mary C. , Backman Lindsey R. F. , Li Phoebe L. , Ulrich Emily C.
Published: May 2022

Glycyl radical enzymes (GREs) employ a post‐translationally installed glycyl radical species to carry out a wide range of chemistry in bacteria. The glycyl radical cofactor is a simple and effective catalyst, but it has an Achilles’ heel – it is susceptible to cleavage by molecular oxygen. For this reason, GREs are commonly expressed in bacteria under anoxic conditions, but not exclusively. The most well studied GRE, pyruvate formate lyase (PFL), is constitutively expressed, and this enzyme acts in primary metabolism, converting pyruvate and coenzyme A into acetyl‐CoA and formate, and is thus not an expendable enzyme. In this presentation, a mechanism for PFL repair from oxidative damage will be presented. In particular, we will consider how a small (14 kDa) ‘spare part protein’ termed YfiD can bind to an oxygen‐damaged PFL (PFL cleaved at Gly734) and restore activity. Our mechanism for repair takes advantage of multiple pieces of data from multiple biochemical and biophysical methods. Through making numerous constructs of both YfiD and PFL, we were able to use enzymology and spectroscopy to determine the regions of each protein that are required for YfiD‐PFL complex formation and activity restoration. We further were able to probe the molecular basis for glycyl radical installation on YfiD and the molecular signal that indicates that PFL is damaged and needs repair but is not too damaged for successful repair. Typically, damaged enzymes are cleared from the cell and new enzymes are made. Here, through use of a spare part protein, the 170‐kDa PFL gets a second chance.
Some assembly required: structural and mechanistic studies of histone chaperones, nucleosome assembly factors and ATP dependent chromatin remodelers
Authors: Luger Karolin , Zhou Keda , Markert Jonathan , Liu Yang
Published: May 2022

All eukaryotes organize their DNA into nucleosomes, consisting of an octamer of the four core histone proteins H2A, H2B, H3, and H4, around which 147 base pairs of DNA are wrapped in two tight superhelical turns. Nucleosomes pack into higher order structures and require histone chaperones and ATP‐dependent remodelers to make the DNA accessible. I will discuss our recent progress in understanding the higher order structure of chromatin at the centromere, and the role of centromere‐binding proteins in locally folding the chromatin fiber. I will also discuss mechanisms by which nucleosomes are remodeled and assembled in an ATP dependent and ATP‐independent manner. The histone chaperone FACT binds and stabilizes partially disassembled nucleosomes by employing an acidic C‐terminal domain as a DNA mimic to clamp down a histone dimer. The chromatin remodeler SMARCAD1 requires ATP hydrolysis to remove histones from a nucleosome, which it then can deposit onto a new DNA segment in an ATP‐independent mechanism, and I will present biochemical and structural evidence for this mode of action.
Sex‐dimorphism in aging: are we missing half of the picture?
Authors: Benayoun Bérénice A. , Lu Ryan J. , Kim Minhoo , McGill Cassandra J.
Published: May 2022

Aging is accompanied by striking changes in chromatin and gene expression across cell types and species. Yet, how chromatin landscapes change with age and regulate transcription, and how epigenomic changes in turn influence aging in response to external or internal cues, is largely unknown. In addition, accumulating evidence indicates that sex hormones play a key role in driving aspects of cellular and molecular sex‐dimorphism. In parallel to sex hormones, karyotypic sex (i.e. XX vs. XY) is also likely to have important impact outside of gonadal sex determination. A key compartment whose activity can be actively modulated by sex‐dimorphic mechanisms throughout life is the immune system, whose function declines sharply with aging and may be actively modulated by sex. Indeed, aspects of the immune responses differ between sexes, with a more robust immune response in females and an increased susceptibility to infection in males. Thus, our main cell models of study are key components of the innate immune system and the inflammatory response: macrophages, which accomplish key tasks such as phagocytosis, antigen presentation and cytokine production, and neutrophils, the most abundant leukocyte type serving as a “first line of defense” against infection. Excitingly, we and others have observed strong sex‐related differences in the transcriptional and functional phenotypes of these cells and have observed sex‐dimorphic “omic” trajectories for these cells with aging. Based on our data and published literature, it is likely that mechanisms involving both gonadal hormones and sex chromosomes may fine‐tune different aspects of immunity and, thus, overall health and lifespan.
The degradation of misfolded proteins in the ER
Authors: Brodsky Jeffrey L.
Published: May 2022

To maintain protein homeostasis (i.e., “proteostasis”) and withstand the toxic effects brought about by the presence of misfolded proteins, eukaryotes have evolved a hierarchy of quality control checkpoints along the secretory pathway. The most prominent quality control step in this pathway, which acts during or soon after proteins are synthesized, is endoplasmic reticulum associated degradation (ERAD). The importance of this pathway is underscored by the fact that ~80 different protein substrates of the ERAD pathway have been linked to human disease. Although most misfolded proteins in the secretory pathway are eliminated by ERAD, others can exit the ER in COPII vesicles and are instead turned over by lysosomal proteases. This post‐ER quality control event requires the ESCRT machinery. A different class of secreted proteins, particularly those that are aggregation‐prone, can alternatively be degraded by ER‐phagy. To date, it remains elusive how these diverse misfolded proteins‐‐which can trigger various stress responses‐‐are selected for different fates. However, by constructing a collection of model substrates and examining wild‐type and disease‐associated mutant forms of various proteins, we are beginning to define the requirements for the targeted selection of misfolded proteins in the secretory pathway for one fate versus another. This pursuit represents a vital step toward the development of pharmaceuticals that might one day repair folding‐defective proteins. Indeed, a growing number of clinical and pre‐clinical drugs that repair ERAD and other quality control substrates have shown efficacy in various disease models. In this presentation, each of these topics will be discussed and future research directions defined.
Common signaling principles and interconnectivity in the ISR‐UPR networks
Authors: Acosta‐Alvear Diego
Published: May 2022

The integrated stress response (ISR) and the unfolded protein response (UPR) are conserved signaling networks governed by stress sensor kinases. Four ISR sensor kinases, GCN2, HRI, PERK, and PKR, phosphorylate eIF2α in response to multiple stresses, leading to a global protein synthesis shutdown coupled to the translation of select mRNAs, which results in an adaptive remodeling of the proteome. Besides PERK, which the ISR and the UPR share, the UPR relies on two additional ER stress sensors, the kinase/RNase IRE1 and the membrane‐tethered transcription factor ATF6, to induce adaptive signaling programs that reinstate ER homeostasis. However, if the stress is unrelenting, both the ISR and the UPR can switch to drive cell death. The mechanistic commonalities and crosstalk between the ISR and UPR remain an active area of investigation. In this talk, I will share new results that support common signaling principles of the ISR and UPR sensor kinases and reveal a new layer of communication between the ISR and the UPR. Specifically, we found that dynamic clustering is a prominent feature of PKR activation reminiscent of the high‐order assemblies of IRE1 and PERK observed during ER stress. Surprisingly, PKR clusters excluded eIF2α, and mutations in PKR that disrupt cluster assembly enhanced eIF2α phosphorylation, suggesting that PKR clusters act as enzyme sinks that control enzyme‐substrate interactions by limiting PKR‐eIF2α encounters. Moreover, stress‐free activation of PKR induced a master cell death program dependent on ISR‐driven expression of DR5 (death receptor 5), as occurs during unmitigable ER stress. Remarkably, stress‐free activation of the ISR selectively activated IRE1 independent of sensing unfolded proteins in the ER lumen, and treatment with the small‐molecule ISR inhibitor ISRIB reversed it. Our data provide new mechanistic insights into two fundamental aspects of stress sensor kinase signaling: (1) dynamic clustering of stress sensors may provide the means to fine‐tune stress responses, and (2) the ISR selectively activates IRE1, thus coupling the ISR and the UPR outside their common node PERK.
Beyond Lysine Ubiquitination
Authors: Virdee Satpal
Published: May 2022

The covalent attachment of ubiquitin to substrates controls virtually all aspects of the cell. With rare exceptions, ubiquitin was understood to exert its effects by becoming attached to the amino group of lysine residues within protein substrates. Recent discoveries from our lab and others have revealed that dedicated writers (E3 ligases) and erasers (DUBs) of non‐lysine ubiquitination are intrinsic to eukaryotes. This highlights that attachment to sites beyond lysine are physiologically and perhaps pathologically important. These E3s tend to be highly divergent from their established counterparts and “activity‐based” chemical biology approaches have been instrumental in identifying them. I will present work from my lab on technologies we have used to uncover unusual E3 ligases and the striking nature of their mechanism and substrate specificity. I will also discuss our characterisation of a small DUB family that is highly selective at removing ubiquitin from hydroxy amino acids. These new developments indicate that non‐lysine ubiquitination is an integral component of the ubiquitin system that is subject to sophisticated regulation.
Novel pathways of intracellular membrane lipid transport and neurodegenerative diseases
Authors: De Camilli Pietro
Published: May 2022

Membrane lipids move from one compartment to another within cells as part of the membranes of vesicular carriers. However, this mode of lipid traffic is complemented by the action of lipid transport proteins that often act at membrane contact sites. Typically, lipid transport by these proteins is achieved by lipid binding modules that shuttle back and forth between the participating membranes. However, recently, studies of VPS13 family proteins (which also comprise the autophagy factor ATG2) have revealed a new mechanism of lipid transfer mediated by rod‐like proteins that bridge two adjacent bilayers and harbor a hydrophobic groove that runs along their entire length. Most interestingly, loss of function mutations of members of this protein family result in neurodegenerative or neurodevelopmental diseases, including Parkinson’s disease (VPS13C) and a Huntington‐like syndrome called chorea acanthocytosis (VPS13A). The talk will provide an overview of the known properties and functions of mammalian VPS13 family proteins and of potential roles of their dysfunction in disease (Leonzino M, Reinisch KM and De Camilli P. 2021. Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport. PMID: 34216812).
Biosynthesis and Function of the Nickel‐Pincer Nucleotide Cofactor
Authors: Hausinger Robert P.
Published: May 2022

Lactate racemase interconverts the L‐ and D‐isomers of lactic acid, a central metabolic intermediate in cells. In 2015, the Hausinger laboratory discovered that the enzyme responsible for this activity in Lactobacillus plantarum, LarA, possesses a covalently‐tethered coenzyme they named the nickel‐pincer nucleotide (NPN) cofactor. This novel organometallic molecule contains a square‐planar nickel atom bound by one histidine residue and tri‐coordinated by a modified pyridinium mononucleotide forming C‐Ni and two S‐Ni bonds. In this seminar, Dr. Hausinger will describe an array of structure/function studies that characterize the three enzymes used for biosynthesis of the NPN cofactor. LarB is a carboxylase/hydrolase of nicotinic acid adenine dinucleotide that forms a dicarboxylated pyridine mononucleotide (P2CMN). LarE is an ATP‐dependent sacrificial sulfur insertase that converts P2CMN into a species with two thiocarboxylic acids (P2TMN). Finally, LarC is a CTP‐dependent nickel insertase or cyclometallase that transforms P2TMN into NPN. The NPN cofactor is incorporated into a variety of 2‐hydroxyacid racemases and selected sugar epimerases where it catalyzes proton‐coupled hydride transfer reactions.