A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

The International Space Station Environment Triggers Molecular Responses in Aspergillus niger.

Due to immense phenotypic plasticity and adaptability, Aspergillus niger is a cosmopolitan fungus that thrives in versatile environments, including the International Space Station (ISS). This is the first report of genomic, proteomic, and metabolomic alterations observed in A. niger strain JSC-093350089 grown in a controlled experiment aboard the ISS. Whole-genome sequencing (WGS) revealed that ISS conditions, including microgravity and enhanced irradiation, triggered non-synonymous point mutations in specific regions, chromosomes VIII and XII of the JSC-093350089 genome when compared to the ground-grown control. Proteome analysis showed altered abundance of proteins involved in carbohydrate metabolism, stress response, and cellular amino acid and protein catabolic processes following growth aboard the ISS. Metabolome analysis further confirmed that space conditions altered molecular suite of ISS-grown A. niger JSC-093350089. After regrowing both strains on Earth, production of antioxidant-Pyranonigrin A was significantly induced in the ISS-flown, but not the ground control strain. In summary, the microgravity and enhanced irradiation triggered unique molecular responses in the A. niger JSC-093350089 suggesting adaptive responses.

Effects of Simulated Microgravity on the Proteome and Secretome of the Polyextremotolerant Black Fungus Knufia chersonesos.

Black fungi are a group of melanotic microfungi characterized by remarkable polyextremotolerance. Due to a broad ecological plasticity and adaptations at the cellular level, it is predicted that they may survive in a variety of extreme environments, including harsh niches on Earth and Mars, and in outer space. However, the molecular mechanisms aiding survival, especially in space, are yet to be fully elucidated. Based on these premises, the rock-inhabiting black fungus Knufia chersonesos (Wt) and its non-melanized mutant (Mut) were exposed to simulated microgravity-one of the prevalent features characterizing space conditions-by growing the cultures in high-aspect-ratio vessels (HARVs). Qualitative and quantitative proteomic analyses were performed on the mycelia and supernatant of culture medium (secretome) to assess alterations in cell physiology in response to low-shear simulated microgravity (LSSMG) and to ultimately evaluate the role of cell-wall melanization in stress survival. Differential expression was observed for proteins involved in carbohydrate and lipid metabolic processes, transport, and ribosome biogenesis and translation via ribosomal translational machinery. However, no evidence of significant activation of stress components or starvation response was detected, except for the scytalone dehydratase, enzyme involved in the synthesis of dihydroxynaphthalene (DNH) melanin, which was found to be upregulated in the secretome of the wild type and downregulated in the mutant. Differences in protein modulation were observed between K. chersonesos Wt and Mut, with several proteins being downregulated under LSSMG in the Mut when compared to the Wt. Lastly, no major morphological alterations were observed following exposure to LSSMG. Similarly, the strains' survivability was not negatively affected. This study is the first to characterize the response to simulated microgravity in black fungi, which might have implications on future astrobiological missions.

PD-L1+ neutrophils contribute to injury-induced infection susceptibility.

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

Proteomics: An advanced tool to unravel the role of alveolar macrophages in respiratory diseases.

As we learn more about chronic lung diseases, we are seeing that an unbalanced immune system plays a key role in disease pathogenesis. Innate immune cells, particularly tissue-resident macrophages, are important navigators of immunity, both during infection and in non-communicable lung disease. In the lung, alveolar macrophages are considered some of the most critical and diverse immune cells, yet despite an array of studies over the years, alveolar macrophages remain poorly understood. In this review, we highlight the importance of alveolar macrophages in health and disease, and discuss how proteomics can be used to elucidate mechanistic information and identify potential targets for therapy development.

Polyunsaturated Fatty Acid Composition of Cerebrospinal Fluid Fractions Shows Their Contribution to Cognitive Resilience of a Pre-symptomatic Alzheimer's Disease Cohort.

Alzheimer's disease (AD) pathology is characterized by an early and prolonged decrease in the amyloid peptide (Aß) levels concomitant with a later increase in phospho-tau concentrations in cerebrospinal fluid (CSF). We propose that changes in lipid metabolism can contribute to the abnormal processing of Aß42 in AD. Our aim was to determine if polyunsaturated fatty acid (PUFA) metabolism can differentiate pre-symptomatic AD from normal aging and symptomatic AD. Using neuropsychology measures and Aß42/T-tau in cerebrospinal fluid (CSF), we classify three groups of elderly study participants: cognitively healthy with normal Aß42/T-tau (CH-NAT), cognitively healthy with pathological Aß42/T-tau (CH-PAT), and AD individuals. We determined the size distribution and the concentration of CSF particles using light scattering and quantified PUFA composition in the nanoparticulate (NP) fraction, supernatant fluid (SF), and unesterified PUFA levels using gas chromatography combined with mass spectrometry. Four PUFAs (C20:2n-6, C20:3n-3, C22:4n-6, C22:5n-3) were enriched in NP of AD compared with CH-NAT. C20:3n-3 levels were higher in the NP fraction from AD compared with CH-PAT. When normalized to the number of NPs in CSF, PUFA levels were significantly higher in CH-NAT and CH-PAT compared with AD. In the SF fractions, only the levels of docosahexaenoic acid (DHA, C22:6n-3) differentiated all three clinical groups. Unesterified DHA was also higher in CH-NAT compared with the other clinical groups. Our studies also show that NP PUFAs in CH participants negatively correlate with CSF Aß42 while C20:4n-6, DHA, and n-3 PUFAs in the SF fraction positively correlate with T-tau. The profile of PUFAs in different CSF fractions that correlate with Aß42 or with T-tau are different for CH-NAT compared with CH-PAT. These studies show that PUFA metabolism is associated with amyloid and tau processing. Importantly, higher PUFA levels in the cognitively healthy study participants with abnormal Aß42/T-tau suggest that PUFA enhances the cognitive resilience of the pre-symptomatic AD population. We propose that interventions that prevent PUFA depletion in the brain may prevent AD pathology by stabilizing Aß42 and tau metabolism. Further studies to determine changes in PUFA composition during the progression from pre-symptomatic to AD should reveal novel biomarkers and potential preventive approaches.

Accumulation of Cerebrospinal Fluid Glycerophospholipids and Sphingolipids in Cognitively Healthy Participants With Alzheimer's Biomarkers Precedes Lipolysis in the Dementia Stage.

Insight into lipids' roles in Alzheimer's disease (AD) pathophysiology is limited because brain membrane lipids have not been characterized in cognitively healthy (CH) individuals. Since age is a significant risk factor of AD, we hypothesize that aging renders the amyloid precursor protein (APP) more susceptible to abnormal processing because of deteriorating membrane lipids. To reflect brain membranes, we studied their lipid components in cerebrospinal fluid (CSF) and brain-derived CSF nanoparticle membranes. Based on CSF Aß42/Tau levels established biomarkers of AD, we define a subset of CH participants with normal Aß42/Tau (CH-NAT) and another group with abnormal or pathological Aß42/Tau (CH-PAT). We report that glycerophospholipids are differentially metabolized in the CSF supernatant fluid and nanoparticle membrane fractions from CH-NAT, CH-PAT, and AD participants. Phosphatidylcholine molecular species from the supernatant fraction of CH-PAT were higher than in the CH-NAT and AD participants. Sphingomyelin levels in the supernatant fraction were lower in the CH-PAT and AD than in the CH-NAT group. The decrease in sphingomyelin corresponded with an increase in ceramide and dihydroceramide and an increase in the ceramide to sphingomyelin ratio in AD. In contrast to the supernatant fraction, sphingomyelin is higher in the nanoparticle fraction from the CH-PAT group, accompanied by lower ceramide and dihydroceramide and a decrease in the ratio of ceramide to sphingomyelin in CH-PAT compared with CH-NAT. On investigating the mechanism for the lipid changes in AD, we observed that phospholipase A2 (PLA2) activity was higher in the AD group than the CH groups. Paradoxically, acid and neutral sphingomyelinase (SMase) activities were lower in AD compared to the CH groups. Considering external influences on lipids, the clinical groups did not differ in their fasting blood lipids or dietary lipids, consistent with the CSF lipid changes originating from brain pathophysiology. The lipid accumulation in a prodromal AD biomarker positive stage identifies perturbation of lipid metabolism and disturbances in APP/Amyloid beta (Aß) as early events in AD pathophysiology. Our results identify increased lipid turnover in CH participants with AD biomarkers, switching to a predominantly lipolytic state in dementia. This knowledge may be useful for targeting and testing new AD treatments.

Alteration of Proteomes in First-Generation Cultures of Bacillus pumilus Spores Exposed to Outer Space.

Bacillus pumilus SAFR-032 was originally isolated from the Jet Propulsion Lab Spacecraft Assembly Facility and thoroughly characterized for its enhanced resistance to UV irradiation and oxidative stress. This unusual resistance of SAFR-032 is of particular concern in the context of planetary protection and calls for development of novel disinfection techniques to prevent extraterrestrial contamination. Previously, spores of SAFR-032 were exposed for 18 months to a variety of space conditions on board the International Space Station to investigate their resistance to Mars-like conditions and space travel. Here, proteomic characterization of vegetative SAFR-032 cells from space-surviving spores is presented in comparison to a ground control. Vegetative cells of the first passage were processed and subjected to quantitative proteomics using tandem mass tags. Approximately 60% of all proteins encoded by SAFR-032 were identified, and 301 proteins were differentially expressed among the strains. We found that proteins predicted to be involved in carbohydrate transport/metabolism and energy production/conversion had lower abundance than those of the ground control. For three proteins, we showed that the expected metabolic activities were decreased, as expected with direct enzymatic assays. This was consistent with a decrease of ATP production in the space-surviving strains. The same space-surviving strains showed increased abundance of proteins related to survival, growth advantage, and stress response. Such alterations in the proteomes provide insights into possible molecular mechanisms of B. pumilus SAFR-032 to adapt to and resist extreme extraterrestrial environments.IMPORTANCE Spore-forming bacteria are well known for their resistance to harsh environments and are of concern for spreading contamination to extraterrestrial bodies during future life detection missions. Bacillus pumilus has been regularly isolated from spacecraft-associated surfaces and exhibited unusual resistance to ultraviolet light and other sterilization techniques. A better understanding of the mechanisms of microbial survival and enhanced resistance is essential for developing novel disinfection protocols for the purpose of planetary protection. While genomic analyses did not reveal the unique characteristics that explain elevated UV resistance of space-exposed B. pumilus, the proteomics study presented here provided intriguing insight on key metabolic changes. The observed proteomics aberrations reveal a complex biological phenomenon that plays a role in bacterial survival and adaptation under long-term exposure to outer space. This adaptive ability of microorganisms needs to be considered by those tasked with eliminating forward contamination.

Lipid Metabolism in Late-Onset Alzheimer's Disease Differs from Patients Presenting with Other Dementia Phenotypes.

Abnormal cerebrospinal fluid (CSF) levels of ß-amyloid peptides (Aß42) and Tau and cognitive decline are typical characteristics of Alzheimer's disease (AD). Since dysregulation in lipid metabolism accompanies abnormal amyloid formation, we quantified glycerophospholipids (GP) and sphingolipids (SP) in CSF fractions from participants with late-onset AD (LOAD, n = 29) or with Other Dementia (OD, n = 10) to determine if alterations in lipid metabolism account for pathological differences. Aß42 and total Tau levels were determined using a sandwich ELISA. Liposomal-based fluorescent assays were used to measure phospholipase A2 (PLA2) and acid or neutral sphingomyelinase (aSMase, nSMase) activities. Supernatant fluid (SF) and nanoparticle (NP) lipids were quantified using LC-MS/MS. Although CSF Aß42 and Tau levels are similar, phosphatidylserine (PS) in SF and ceramide (CM) levels in NP are significantly higher in OD compared with LOAD. The aSMase but not the nSMase activity is higher in OD. PLA2 activity in CSF from OD subjects positively correlates with several GP classes in SF and NP fractions but not in LOAD fractions. Our data indicate differences in CSF lipid metabolism between dementia variants. Higher levels of inflammatory and apoptotic lipids may induce faster neuronal death, resulting in the earlier cognitive decline in patients with OD phenotypes.

Proteomic and Metabolomic Characteristics of Extremophilic Fungi Under Simulated Mars Conditions.

Filamentous fungi have been associated with extreme habitats, including nuclear power plant accident sites and the International Space Station (ISS). Due to their immense adaptation and phenotypic plasticity capacities, fungi may thrive in what seems like uninhabitable niches. This study is the first report of fungal survival after exposure of monolayers of conidia to simulated Mars conditions (SMC). Conidia of several Chernobyl nuclear accident-associated and ISS-isolated strains were tested for UV-C and SMC sensitivity, which resulted in strain-dependent survival. Strains surviving exposure to SMC for 30 min, ISSFT-021-30 and IMV 00236-30, were further characterized for proteomic, and metabolomic changes. Differential expression of proteins involved in ribosome biogenesis, translation, and carbohydrate metabolic processes was observed. No significant metabolome alterations were revealed. Lastly, ISSFT-021-30 conidia re-exposed to UV-C exhibited enhanced UV-C resistance when compared to the conidia of unexposed ISSFT-021.

Proteomic characterization of Aspergillus fumigatus isolated from air and surfaces of the International Space Station.

The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suite of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, respectively, is presented, along with a comparison to well-studied clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.

Two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

International Space Station conditions alter genomics, proteomics, and metabolomics in Aspergillus nidulans.

The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.

Characterization of Aspergillus niger Isolated from the International Space Station.

The initial characterization of the Aspergillus niger isolate JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS), is reported, along with a comparison to the extensively studied strain ATCC 1015. Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement within the A. niger/welwitschiae/lacticoffeatus clade and revealed that the genome of JSC-093350089 is within the observed genetic variance of other sequenced A. niger strains. The ISS isolate exhibited an increased rate of growth and pigment distribution compared to a terrestrial strain. Analysis of the isolate's proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger on board the ISS and provide insight into the characteristics of melanized fungal species inhabiting spacecraft environments. IMPORTANCE A thorough understanding of how fungi respond and adapt to the various stimuli encountered during spaceflight presents many economic benefits and is imperative for the health of crew. As A. niger is a predominant ISS isolate frequently detected in built environments, studies of A. niger strains inhabiting closed systems may reveal information fundamental to the success of long-duration space missions. This investigation provides valuable insights into the adaptive mechanisms of fungi in extreme environments as well as countermeasures to eradicate unfavorable microbes. Further, it enhances understanding of host-microbe interactions in closed systems, which can help NASA's Human Research Program maintain a habitat healthy for crew during long-term manned space missions.

Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection.

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV's beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms.