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We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30–90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org . The meltome atlas compiles the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans, providing a resource for analyzing protein stability in the context of function and interactions.

Lipid species patterns are conserved within cells to maintain physicochemical properties of membranes and cellular functions. We present the lipidome, including sterols, glycerolipids (GLs), glycerophospholipids (GPLs), and sphingolipids (SLs), of primary ex vivo differentiated (I) white, (II) brite, and (III) brown adipocytes derived from primary preadipocytes isolated from (I) epididymal white, (II) inguinal white, and (III) intrascapular brown adipose tissue. Quantitative lipidomics revealed significantly decreased fractions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), with longer (C > 36) and more polyunsaturated species, as well as lower levels of cardiolipin (CL) in white than in brite and brown adipocytes. Together, the brite and brown lipidome was comparable and indicates differences in membrane lipid packing density compared with white adipocytes. Changes in ceramide species profile could be related to the degree of browning. Beta-adrenergic stimulation of brown adipocytes led to generation of saturated lyso-PC (LPC) increasing uncoupling protein (UCP) 1-mediated leak respiration. Application of stable isotope labeling showed that LPC formation was balanced by an increased de novo synthesis of PC.

The regular overconsumption of energy‐dense foods (rich in lipids and sugars) results in elevated intestinal nutrient absorption and consequently excessive accumulation of lipids in the liver, adipose tissue, skeletal muscles, and other organs. This can eventually lead to obesity and obesity‐associated diseases such as type 2 diabetes (T2D), non‐alcoholic fatty liver disease (NAFLD), cardiovascular disease, and certain types of cancer, as well as aggravate inflammatory bowel disease (IBD). Therefore, targeting the pathways that regulate intestinal nutrient absorption holds significant therapeutic potential. In this review, we discuss the molecular and cellular mechanisms controlling intestinal lipid handling, their relevance to the development of metabolic diseases, and emerging therapeutic strategies. Graphical Abstract In this review, M. Wit & colleagues discuss the molecular and cellular mechanisms controlling intestinal lipid handling, their relevance to the development of metabolic diseases, as well as emerging therapeutic strategies for the treatment of obesity and obesity‐associated diseases.

Oxidative stress plays a central role in numerous conditions, including cancer, cardiovascular and neurodegenerative diseases, diabetes, chronic inflammation, and organ transplantation. In transplantation, oxidative stress leads to mitochondrial dysfunction, DNA and protein damage, lipid peroxidation, and activation of pro-inflammatory pathways such as NF-κB, ultimately impairing cell viability and organ function. A Kinase-Interacting Protein 1 (AKIP1) has been linked to oxidative stress regulation in transgenic mouse models. To investigate this further in a livestock setting, we generated AKIP1 transgenic pigs and assessed AKIP1’s protective role against oxidative-stress-induced cell death, including apoptosis, necrosis, and ferroptosis in vitro. Our cellular analyses revealed reduced apoptosis (caspase-3/7 activity), suppressed MPTP-mediated necrosis, and decreased lipid peroxidation, suggesting protection from ferroptosis. Additionally, we observed lower mitochondrial superoxide production and enhanced mitochondrial respiration and recovery following H2O2-induced oxidative challenge. This is the first study to examine AKIP1 in porcine cells, providing a unique and translational platform for studying oxidative injury in a physiologically relevant species. Our in vitro data reveal that AKIP1 overexpression enhances antioxidant defenses and mitochondrial stability, offering future potential for improving graft survival in xenotransplantation.

The traditional nomenclature of enteroendocrine cells (EECs), established in 1977, applied the “one cell - one hormone” dogma, which distinguishes subpopulations based on the secretion of a specific hormone. These hormone-specific subpopulations included S cells for secretin (SCT), K cells for glucose-dependent insulinotropic polypeptide (GIP), N cells producing neurotensin (NTS), I cells producing cholecystokinin (CCK), D cells producing somatostatin (SST), and others. In the past 15 years, reinvestigations into murine and human organoid-derived EECs, however, strongly questioned this dogma and established that certain EECs coexpress multiple hormones. Using the Gut Cell Atlas, the largest available single-cell transcriptome dataset of human intestinal cells, this study consolidates that the original dogma is outdated not only for murine and human organoid-derived EECs, but also for primary human EECs, showing that the expression of certain hormones is not restricted to their designated cell type. Moreover, specific analyses into SCT-expressing cells reject the presence of any cell population that exhibits significantly elevated secretin expression compared to other cell populations, previously referred to as S cells. Instead, this investigation indicates that secretin production is realized jointly by other enteroendocrine subpopulations, validating corresponding observations in murine EECs also for human EECs. Furthermore, our findings corroborate that SCT expression peaks in mature EECs, in contrast, progenitor EECs exhibit markedly lower expression levels, supporting the hypothesis that SCT expression is a hallmark of EEC maturation.

Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Here we report that mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using intestinal epithelial cell (IEC)-specific mouse models, we show that loss of HSP60, a mitochondrial chaperone, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction. HSP60-deficient crypts display loss of stemness and cell proliferation, accompanied by epithelial release of WNT10A and RSPO1. Sporadic failure of Cre-mediated Hsp60 deletion gives rise to hyperproliferative crypt foci originating from OLFM4 + stem cells. These effects are independent of the MT-UPR-associated transcription factor CHOP. In conclusion, compensatory hyperproliferation of HSP60 + escaper stem cells suggests paracrine release of WNT-related factors from HSP60-deficient, functionally impaired IEC to be pivotal in the control of the proliferative capacity of the stem cell niche. It is unclear what role mitochondrial function plays in maintaining intestinal epithelial cell (IEC) homeostasis. Here, the authors deplete a mitochondrial chaperone, heat shock protein 60 (HSP60) in IEC and observe a loss of stemness and cell proliferation, and suggest this is accompanied by a compensatory release of WNT-related factors.

Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic ( R / S )-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, ( R / S )-lipoic acid is an approved drug for the treatment of diabetic neuropathy. However, its mechanism of action remains elusive. Here, we performed chemoproteomics-aided target deconvolution of lipoic acid and its active close analog lipoamide. We find that histone deacetylases HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC10 are molecular targets of the reduced form of lipoic acid and lipoamide. Importantly, only the naturally occurring ( R )-enantiomer inhibits HDACs at physiologically relevant concentrations and leads to hyperacetylation of HDAC substrates. The inhibition of HDACs by ( R )-lipoic acid and lipoamide explain why both compounds prevent stress granule formation in cells and may also provide a molecular rationale for many other phenotypic effects elicited by lipoic acid. Lipoic acid is a food supplement and approved neuropathy drug but lacks known molecular targets. Here, Lechner et al . employ chemoproteomic target deconvolution to demonstrate that ( R )- but not ( S )-lipoic acid stereoselectively inhibits histone deacetylases at physiologically achievable doses.

During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α . The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62 Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62 Δ69-251 mice, global p62 −/− and Ucp1-Cre p62 flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding. Beta-adrenergic stimulation of brown adipose tissue leads to thermogenesis via the activating transcription factor 2 (ATF2) mediated expression of the thermogenic genes Ucp1 and Pgc-1α . Here, the authors show that the scaffold protein p62 regulates brown adipose tissue function through modifying ATF2 genomic binding and subsequent Ucp1 and Pgc-1α induction.

Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.

Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn's disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn's disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn's disease-like ileitis. TNF(ΔARE/WT) mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors. HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNF(ΔARE/WT). Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria. HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn's disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.