The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4β-hydroxycholesterol (4βHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6β-epoxycholesterol (5,6βEC), 0.51; cholesterol, 0.70; cholestane-3β,5α,6β-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6βEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4βHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4βHC. Substantial FA esterification of 4βHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.

TG-rich lipoprotein (TRL)-related biomarkers, including TRL-cholesterol (TRL-C), remnant-like lipoprotein particle-cholesterol (RLP-C), and apoC-III have been associated with atherosclerosis. However, their prognostic values have not been fully determined, especially in patients with previous CAD. This study aimed to examine the associations of TRL-C, RLP-C, and apoC-III with incident cardiovascular events (CVEs) in the setting of secondary prevention of CAD. Plasma TRL-C, RLP-C, and total apoC-III were directly measured. A total of 4,355 participants with angiographically confirmed CAD were followed up for the occurrence of CVEs. During a median follow-up period of 5.1 years (interquartile range: 3.9–6.4 years), 543 (12.5%) events occurred. Patients with incident CVEs had significantly higher levels of TRL-C, RLP-C, and apoC-III than those without events. Multivariable Cox analysis indicated that a log unit increase in TRL-C, RLP-C, and apoC-III increased the risk of CVEs by 49% (95% CI: 1.16–1.93), 21% (95% CI: 1.09–1.35), and 40% (95% CI: 1.11–1.77), respectively. High TRL-C, RLP-C, and apoC-III were also independent predictors of CVEs in individuals with LDL-C levels ≤1.8 mmol/l (n = 1,068). The addition of RLP-C level to a prediction model resulted in a significant increase in discrimination, and all three TRL biomarkers improved risk reclassification. Thus, TRL-C, RLP-C, and apoC-III levels were independently associated with incident CVEs in Chinese CAD patients undergoing statin therapy.

For three decades, the LPL–specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Accompanied with nutrition transition, non-HDL-C levels of individuals in Asian countries has increased rapidly, which has caused the global epicenter of nonoptimal cholesterol to shift from Western countries to Asian countries. Thus, it is critical to underline major genetic and dietary determinants. In the current study of 2,330 Chinese individuals, genetic risk scores (GRSs) were calculated for total cholesterol (TC; GRS_TC, 57 SNPs), LDL-C (GRS_LDL-C, 45 SNPs), and HDL-C (GRS_HDL-C, 65 SNPs) based on SNPs from the Global Lipid Genetics Consortium study. Cholesterol intake was estimated by a 74-item food-frequency questionnaire. Associations of dietary cholesterol intake with plasma TC and LDL-C strengthened across quartiles of the GRS_TC (effect sizes: –0.29, 0.34, 2.45, and 6.47; P_interaction = 0.002) and GRS_LDL-C (effect sizes: –1.35, 0.17, 5.45, and 6.07; P_interaction = 0.001), respectively. Similar interactions with non-HDL-C were observed between dietary cholesterol and GRS_TC (P_interaction = 0.001) and GRS_LDL-C (P_interaction = 0.004). The adverse effects of GRS_TC on TC (effect sizes across dietary cholesterol quartiles: 0.51, 0.82, 1.21, and 1.31; P_interaction = 0.023) and GRS_LDL-C on LDL-C (effect sizes across dietary cholesterol quartiles: 0.66, 0.52, 1.12, and 1.56; P_interaction = 0.020) were more profound in those having higher cholesterol intake compared with those with lower intake. Our findings suggest significant interactions between genetic susceptibility and dietary cholesterol intake on plasma cholesterol profiles in a Chinese population.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-B via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS³). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Human genetic studies recently identified an association of SNPs in the 17-β hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/β-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC.

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^–/– mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

The organic anion transporters (OATs) and organic anion–transporting polypeptides (OATPs) belong to the solute carrier (SLC) transporter superfamily and play important roles in handling various endogenous and exogenous compounds of anionic charge. The OATs and OATPs are often implicated in drug therapy by impacting the pharmacokinetics of clinically important drugs and, thereby, drug exposure in the target organs or cells. Various mechanisms (e.g. genetic, environmental, and disease-related factors, drug-drug interactions, and food-drug interactions) can lead to variations in the expression and activity of the anion drug-transporting proteins of OATs and OATPs, possibly impacting the therapeutic outcomes. Previous investigations mainly focused on the regulation at the transcriptional level and drug-drug interactions as competing substrates or inhibitors. Recently, evidence has accumulated that cellular trafficking, post-translational modification, and degradation mechanisms serve as another important layer for the mechanisms underlying the variations in the OATs and OATPs. This review will provide a brief overview of the major OATs and OATPs implicated in drug therapy and summarize recent progress in our understanding of the post-translational modifications, in particular ubiquitination and degradation pathways of the individual OATs and OATPs implicated in drug therapy.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC–MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G₁. Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1^WT versus Kir2.1^314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 10³ HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is used in hospitals for VAP diagnosis and guiding treatment options. Although BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their use has not been thoroughly explored. Longitudinal ETA and BAL were collected from 16 intubated patients up to 15 days, of which 11 developed VAP. We conducted a comprehensive LC–MS/MS based proteome and metabolome characterization of longitudinal ETA and BAL to detect host and pathogen responses to VAP infection. We discovered a diverse ETA proteome of the upper airways reflective of a rich and dynamic host-microbe interface. Prior to VAP diagnosis by microbial cultures from BAL, patient ETA presented characteristic signatures of reactive oxygen species and neutrophil degranulation, indicative of neutrophil mediated pathogen processing as a key host response to the VAP infection. Along with an increase in amino acids, this is suggestive of extracellular membrane degradation resulting from proteolytic activity of neutrophil proteases. The metaproteome approach successfully allowed simultaneous detection of pathogen peptides in patients' ETA, which may have potential use in diagnosis. Our findings suggest that ETA may facilitate early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore provide its diagnosis and treatment.

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Ų and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

Natural compounds that can stimulate salivary secretion are of interest in developing treatments for xerostomia, the perception of a dry mouth, that affects between 10 and 30% of the adult and elderly population. Chemesthetic transient receptor potential (TRP) channels are expressed in the surface of the oral mucosa. The TRPV1 agonists capsaicin and piperine have been shown to increase salivary flow when introduced into the oral cavity but the sialogogic properties of other TRP channel agonists have not been investigated. In this study we have determined the influence of different TRP channel agonists on the flow and protein composition of saliva. Mouth rinsing with the TRPV1 agonist nonivamide or menthol, a TRPM8 agonist, increased whole mouth saliva (WMS) flow and total protein secretion compared with unstimulated saliva, the vehicle control mouth rinse or cinnamaldehyde, a TRPA1 agonist. Nonivamide also increased the flow of labial minor gland saliva but parotid saliva flow rate was not increased. The influence of TRP channel agonists on the composition and function of the salivary proteome was investigated using a multi-batch quantitative MS method novel to salivary proteomics. Inter-personal and inter-mouth rinse variation was observed in the secreted proteomes and, using a novel bioinformatics method, inter-day variation was identified with some of the mouth rinses. Significant changes in specific salivary proteins were identified after all mouth rinses. In the case of nonivamide, these changes were attributed to functional shifts in the WMS secreted, primarily the over representation of salivary and nonsalivary cystatins which was confirmed by immunoassay. This study provides new evidence of the impact of TRP channel agonists on the salivary proteome and the stimulation of salivary secretion by a TRPM8 channel agonist, which suggests that TRP channel agonists are potential candidates for developing treatments for sufferers of xerostomia.

The neuronal basis of complex social behavior is still poorly understood. In honeybees, reproductive investment decisions are made at the colony-level. Queens develop from female-destined larvae that receive alloparental care from nurse bees in the form of ad-libitum royal jelly (RJ) secretions. Typically, the number of raised new queens is limited but genetic breeding of "royal jelly bees" (RJBs) for enhanced RJ production over decades has led to a dramatic increase of reproductive investment in queens. Here, we compare RJBs to unselected Italian bees (ITBs) to investigate how their cognitive processing of larval signals in the mushroom bodies (MBs) and antennal lobes (ALs) may contribute to their behavioral differences. A cross-fostering experiment confirms that the RJB syndrome is mainly due to a shift in nurse bee alloparental care behavior. Using olfactory conditioning of the proboscis extension reflex, we show that the RJB nurses spontaneously respond more often to larval odors compared with ITB nurses but their subsequent learning occurs at similar rates. These phenotypic findings are corroborated by our demonstration that the proteome of the brain, particularly of the ALs differs between RJBs and ITBs. Notably, in the ALs of RJB newly emerged bees and nurses compared with ITBs, processes of energy and nutrient metabolism, signal transduction are up-regulated, priming the ALs for receiving and processing the brood signals from the antennae. Moreover, highly abundant major royal jelly proteins and hexamerins in RJBs compared with ITBs during early life when the nervous system still develops suggest crucial new neurobiological roles for these well-characterized proteins. Altogether, our findings reveal that RJBs have evolved a strong olfactory response to larvae, enabled by numerous neurophysiological adaptations that increase the nurse bees' alloparental care behavior.

Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. We then showed that long, but not short, exposure to Interferon- induces a similar signature in vitro. We identified 10 different temporal protein expression patterns, classifying the Interferon- protein temporal regulation in CRC. Our data sheds light on the changes that tumor cells undergo under long-term immunological pressure in vivo, the importance of STAT proteins in specific biological scenarios. The data generated could help find novel clinical biomarkers and therapeutic approaches.

A key point in achieving accurate intact glycopeptide identification is the definition of the glycan composition file that is used to match experimental with theoretical masses by a glycoproteomics search engine. At present, these files are mainly built from searching the literature and/or querying data sources focused on posttranslational modifications. Most glycoproteomics search engines include a default composition file that is readily used when processing MS data. We introduce here a glycan composition visualizing and comparative tool associated with the GlyConnect database and called GlyConnect Compozitor. It offers a web interface through which the database can be queried to bring out contextual information relative to a set of glycan compositions. The tool takes advantage of compositions being related to one another through shared monosaccharide counts and outputs interactive graphs summarizing information searched in the database. These results provide a guide for selecting or deselecting compositions in a file in order to reflect the context of a study as closely as possible. They also confirm the consistency of a set of compositions based on the content of the GlyConnect database. As part of the tool collection of the Glycomics@ExPASy initiative, Compozitor is hosted at https://glyconnect.expasy.org/compozitor/ where it can be run as a web application. It is also directly accessible from the GlyConnect database.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.