Fragile X syndrome (FXS) is a monogenic neurodevelopmental disorder with molecular, neuroanatomical, and behavioral changes. In FXS, astrocytes express dysregulated gene and protein networks, therefore identifying upstream pathways mediating astrocyte changes may provide a point of intervention. We focus on the bone morphogenetic protein (BMP) pathway, which is upregulated in FXS astrocytes. We generated a conditional KO (cKO) of Smad4 in astrocytes to suppress BMP signaling, finding that this lessens audiogenic seizure severity in male FXS mice. We performed in vivo transcriptomic and proteomic profiling of cortical astrocytes, finding upregulation of metabolic pathways, and downregulation of secretory machinery and secreted and membrane proteins in FXS astrocytes, with these alterations mitigated when BMP signaling is suppressed. Functionally, astrocyte Smad4 cKO restores deficits in inhibitory synapses in the FXS auditory cortex. Our findings show that astrocytes contribute to some FXS mouse molecular and functional phenotypes, and targeting astrocyte BMP signaling improves some FXS symptoms.

Trifluoroacetate (TFA) has been assumed to be an innocuous counterion in countless synthetic bioactive peptides and a few FDA-approved therapeutics. Here we show that TFA is in fact bioactive, inducing peroxisome proliferation and causing significant metabolic effects in three strains of mice (wild-type C57BL/6 J, Ldlr−/−, and Apoe−/−) and cultured liver cells. In a high-fat diet induced Ldlr−/− mouse model of atherosclerosis, TFA reduced the levels of plasma LDL cholesterol, triglycerides, and the development of atherosclerotic lesions. These physiological effects were observed with TFA alone, or with TFA present as a counterion of a variety of short, unrelated synthetic peptide sequences. Multiple lines of mechanistic evidence supported that TFA acts by binding to peroxisome proliferator activated receptor (PPAR)-α to induce peroxisome proliferation. These findings indicate the need to assess the contribution of TFA to phenotypic effects observed in some studies using synthetic peptides and, if these effects extend to humans, suggest potential therapeutic applications as well as broader health implications of widespread environmental TFA exposure. Trifluoroacetate (TFA), long assumed to be an inert peptide counterion, is a bioactive modulator of PPARα signaling that induces peroxisome proliferation, alters lipid metabolism, and attenuates atherosclerosis in mice.

Partial lipodystrophy is characterized by abnormal fat distribution, impaired thermogenesis, systemic inflammation, and diminished DICER1 expression in adipose tissue. Using adipocyte-specific Dicer1-deficient murine models to mimic lipodystrophy, we identified early metabolic and inflammatory alterations associated with the onset of partial lipodystrophy. DICER deficiency in adipocytes impaired lipid metabolism, leading to increased release of saturated lipids and triglycerides into the medium while reducing the biosynthesis of anti-inflammatory lipids. These changes promoted a pro-inflammatory environment by inducing macrophages to release IL-1β. In parallel, both adipocyte-autonomous DICER deficiency and the resulting inflammatory environment impaired adipocyte thermogenic capacity. Lipidomic analyses revealed reduced levels of docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) in Dicer1-deficient adipocytes, accompanied by increased expression of genes encoding CYP450 enzymes linked to lipid degradation, and reduced DHA biosynthesis. Co-culture experiments confirmed that Dicer1-deficient adipocytes exacerbate macrophage-driven pro-inflammatory profile and thermogenic gene suppression. Notably, DHA supplementation mitigated the pro-inflammatory response triggered by conditioned media from Dicer1-deficient adipocytes. These findings reveal a critical role for adipocyte DICER in regulating lipid metabolism, macrophage-adipocyte interactions, and thermogenic function, offering new insights into molecular mechanisms driving metabolic dysfunction and chronic inflammation associated with partial lipodystrophy.

To understand the mechanism of action of a drug and assess its clinical usefulness and viability, it is imperative that its affinity for its putative targets is determined. When coupled to mass spectrometry (MS), energetics-based protein separation (EBPS) techniques, such as a thermal shift assay, have shown great potential to identify the targets of a drug on a proteome scale. Nevertheless, the computational analyses assessing the confidence of drug-target predictions made by these methods have remained tightly tied to the protocol under which the data were produced. To identify drug targets in data sets produced using different EBPS-MS techniques, we have developed a novel flexible Bayesian inference approach named TargetSeeker-MS. We showed that TargetSeeker-MS identifies known and novel drug targets in Caenorhabditis elegans and HEK 293 samples treated with the fungicide benomyl. We also demonstrated that TargetSeeker-MS' drug-target identifications are reproducible in C. elegans samples that were processed using two different EBPS techniques (thermal shift assay and a differential precipitation of proteins, named DiffPOP). In addition, we validated a novel benomyl target by measuring its altered enzymatic activity upon drug treatment in vitro. TargetSeeker-MS, which is available as a web server (https://targetseeker.scripps.edu/), allows for the rapid, versatile, and confident identification of targets of a drug on a proteome scale, thereby providing a better understanding of its mechanisms and facilitating the evaluation of its clinical viability.

Aging is associated with dysregulated methionine metabolism and increased levels of enzymes in the tyrosine degradation pathway (TDP). To investigate the efficacy of targeting either methionine metabolism or the TDP for healthspan improvement in advanced age, we initiated dietary MetR or TDP inhibition in 18-month-old C57BL/6J mice. MetR significantly improved neuromuscular function, metabolic health, lung function, and frailty. In addition, we confirmed improved neuromuscular function from dietary MetR in 5XFAD mice, whose weight was not affected by MetR. We did not observe benefits with TDP inhibition. Single-nucleus RNA and ATAC sequencing of muscle revealed cell type-specific responses to MetR, although MetR did not significantly affect mouse aging epigenetic clock markers. Similarly, an 8-week MetR intervention in a human trial (NCT04701346) showed no significant impact on epigenetic clocks. The observed benefits from late-life MetR provide translational rationale to develop MetR mimetics as an antiaging intervention.

Membrane frizzled-related protein (MFRP), present in the retinal pigment epithelium (RPE), is an integral membrane protein essential for ocular development and the normal physiology of the retina. Mutations in MFRP are associated with autosomal recessive nonsyndromic nanophthalmos, leading to severe hyperopia and early-onset retinitis pigmentosa. While several preclinical gene-augmentation and gene-editing trials hold promise for future therapies aimed at stopping degeneration and restoring retinal function, the molecular mechanisms involved in MFRP biology are still not well understood. Here, we studied the biochemical properties of MFRP and the molecular consequences of its loss of function in the retinal degeneration 6 (rd6) mouse model. Using transcriptomic and lipidomic approaches, we observed that accumulation of docosahexaenoic acid (DHA) constitutes a primary defect in the MFRP-deficient RPE. In biochemical assays, we showed that MFRP undergoes extensive glycosylation, and it preferentially binds lipids of several classes, including phosphatidylserine and phosphatidylinositol-4-phosphate; as well as binding to several transmembrane proteins, notably adiponectin receptor 1 (ADIPOR1) and inward rectifier potassium channel 13 (KCNJ13). Moreover, MFRP determines the subcellular localization of ADIPOR1 and KCNJ13 in the RPE in vivo. This feature is altered by MFRP deficiency and can be restored by gene-therapy approaches. Overall, our observations suggest that MFRP constitutes an important interaction hub within the apical membrane of RPE cells, coordinating protein trafficking and subcellular localization within the RPE, and lipid homeostasis within the entire retina.

Transthyretin (TTR) variant (V30M) polyneuropathy (ATTRv-PN) is a progressive systemic amyloidosis caused by transthyretin aggregation, leading to a variety of debilitating manifestations, including neuropathy and cardiomyopathy. We investigated the plasma proteome of heterozygotic V30M TTR asymptomatic carriers and heterozygotic V30M ATTRv-PN patients (before and after tafamidis treatment) versus WT TTR healthy control plasma using an organic solvent-induced shift in solubility assay to identify biosignatures for disease progression and therapeutic response. We identified many proteins, including TTR, apolipoproteins, ceruloplasmin, and proteins with functions in innate immunity that displayed changes in either their abundances or their sensitivity to precipitation. Elevated oxidative modifications of TTR and APOE in ATTRv-PN patients suggest a role for oxidative stress in disease pathogenesis/progression. Tafamidis treatment mitigated these pathology-associated changes, suggesting that alleviating proteotoxic stress impacts these other pathways. Although our study was limited to a Portuguese cohort, these findings nevertheless provide a comprehensive plasma proteomic profile of V30M ATTRv-PN patients, V30M TTR carriers, and tafamidis-treated ATTRv-PN patients over up to 60 months; provide insights into ATTRv-PN pathophysiology; identify potential biomarkers for disease progression and therapeutic response; and highlight the utility of proteomics in advancing personalized treatments for amyloidosis.

Trifluoroacetate (TFA) has been assumed to be an innocuous counterion (to cationic amino acid side chains) present in countless synthetic bioactive peptides and a few FDA-approved therapeutics. We show here that TFA is in fact bioactive and causes dramatic biological effects in multiple strains of mice and cultured human and rat liver cells. In high-fat diet (HFD)-fed low-density lipoprotein receptor-null (LDLr-/-) mice, TFA reduces the levels of plasma cholesterol, triglycerides, and the development of atherosclerotic lesions following either oral or intraperitoneal administration. These physiological effects were observed with TFA alone, or with TFA present as a counterion of a variety of short, unrelated synthetic peptide sequences. Mechanistic investigations including RNA-seq, confocal microscopy, western blotting, metabolomics, proteomics, pharmacokinetics, and biochemical assays indicated that TFA induces peroxisome proliferation by activating peroxisome proliferator-activated receptor (PPAR)-alpha. We confirmed that TFA also caused peroxisome proliferation and downstream phenotypic effects in cultured human and rat liver cells, wild-type C57/Bl mice, and apolipoprotein E-null (apoE-/-) mice, leading to anti-atherosclerotic effects in the latter strain. Given that TFA is a counterion in many peptides employed in early research and development settings, these findings raise the possibility that TFA may be confounding or contributing to phenotypic changes observed in many studies involving peptides. Although our studies suggest that TFA or its analogues might have therapeutic applications, it should be noted that TFA is also a persistent environmental contaminant that is found at high levels in humans relative to other polyfluoroalkyl substances (PFAS), and is a major metabolite following treatment of patients with common inhaled anesthetics, suggesting that the biological effects reported here could have other implications for human health. ### Competing Interest Statement The authors have declared no competing interest.

Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction of mice using ribosome profiling. Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 single guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. The smORFs on the 5'- and 3'-untranslated regions (i.e., upstream smORFs (uORFs) and downstream smORFs (dORFs)) of functional genes can exert activity through cis-regulatory effects of the main ORF on these messenger RNAs (mRNAs), such as uORFs of MDM2 that impact proliferation. However, other smORFs, especially those from mRNAs with no other ORFs, point to a functional microprotein. Indeed, we tested a candidate smORF 1183 from a long noncoding RNA 923011K14Rik and demonstrated that the microprotein regulates adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids and fatty acid hydroxyoctadecadienoic acids in white adipose tissue depots and in adipocytes cocultured with macrophages. In lipopolysaccharide (LPS)-induced coculture systems, we confirm that adipose triglyceride lipase is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized fatty acid hydroxyoctadecadienoic acids show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of proinflammatory hydroxy fatty acids into FAHFAs may dampen inflammation. Indeed, we demonstrate that proinflammatory lipids, such as hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4), can be converted by cells in culture to weakly antiinflammatory FAHFAs.

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids (FAHSAs) and fatty acid hydroxyoctadecadienoic acids (FAHODEs) in white adipose tissue depots and in adipocytes co-cultured with macrophages. In LPS-induced co-culture systems, we confirm that adipose triglyceride lipase (ATGL) is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized FAHODEs show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of pro-inflammatory hydroxy fatty acids into FAHFAs may modulate inflammation. We test this concept by showing the pro-inflammatory lipids—hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4)—are converted into FAHFAs in cell culture, and that two LTB4-derived FAHFAs have are modestly anti- not pro-inflammatory. Further research is needed to establish whether these increased FAFHA levels have a role in inflammation or are simply markers of inflammation, but the discovery of significant increases in FAHFA upon acute inflammation advances our knowledge of FAHFAs.

BACKGROUND:Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS:We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS:TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating β-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS:In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.

Enterococcus faecalis is a resident of the human gut, though upon translocation to the blood or body tissues, it can be pathogenic. Here we discover and characterize two peptide-based quorum-sensing systems that transcriptionally modulate de novo purine biosynthesis in E. faecalis. Using a comparative genomic analysis, we find that most enterococcal species do not encode this system; E. moraviensis, E. haemoperoxidus and E. caccae, three species that are closely related to E. faecalis, encode one of the two systems, and only E. faecalis encodes both systems. We show that these systems are important for the intracellular survival of E. faecalis within macrophages and for the fitness of E. faecalis in a murine wound infection model. Taken together, we combine comparative genomics, microbiological, bacterial genetics, transcriptomics, targeted proteomics and animal model experiments to describe a paired quorum sensing mechanism that directly influences central metabolism and impacts the pathogenicity of E. faecalis.

Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.

Fragile X syndrome (FXS) is a monogenic neurodevelopmental disorder with manifestations spanning molecular, neuroanatomical, and behavioral changes. Astrocytes contribute to FXS pathogenesis and show hundreds of dysregulated genes and proteins; targeting upstream pathways mediating astrocyte changes in FXS could therefore be a point of intervention. To address this, we focused on the bone morphogenetic protein (BMP) pathway, which is upregulated in FXS astrocytes. We generated a conditional KO (cKO) of Smad4 in astrocytes to suppress BMP signaling, and found this lessens audiogenic seizure severity in FXS mice. To ask how this occurs on a molecular level, we performed in vivo transcriptomic and proteomic profiling of cortical astrocytes, finding upregulation of metabolic pathways, and downregulation of secretory machinery and secreted proteins in FXS astrocytes, with these alterations no longer present when BMP signaling is suppressed. Functionally, astrocyte Smad4 cKO restores deficits in inhibitory synapses present in FXS auditory cortex. Thus, astrocytes contribute to FXS molecular and functional phenotypes, and targeting astrocytes can mitigate FXS symptoms.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration (AMD). This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 FAs, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α (PPARα) signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium (RPE). Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome (BBS) proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in BBS-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including AMD.

Background When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. Methods Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. Results Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. Conclusions Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods. Graphical Abstract

Differential precipitation of proteins (DiffPOP) is a simple technique for fractionating complex protein mixtures. Using stepwise addition of acidified methanol, ten distinct subsets of proteins can be selectively precipitated by centrifugation and identified by mass spectrometry-based proteomics. We have previously shown that the ability of a protein to resist precipitation can be altered by drug binding, which enabled us to identify a novel drug-target interaction. Here, we show that the addition of DiffPOP to a standard LC-MS proteomics workflow results in a three-dimensional separation of peptides that increases protein coverage and peptide identifications. Importantly, DiffPOP reveals solubility differences between proteoforms, potentially providing valuable insights that are typically lost in bottom-up proteomics.