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Background Birthweight reflects in utero exposures and later health evolution. Despite existing studies employing high-dimensional molecular measurements, the understanding of underlying mechanisms of birthweight remains limited. Methods To investigate the systems biology of birthweight, we cross-sectionally integrated the methylome, the transcriptome, the metabolome and a set of inflammatory proteins measured in cord blood samples, collected from four birth-cohorts (n = 489). We focused on two sets of 68 metabolites and 903 CpGs previously related to birthweight and investigated the correlation structures existing between these two sets and all other omic features via bipartite Pearson correlations. Results This dataset revealed that the set of metabolome and methylome signatures of birthweight have seven signals in common, including three metabolites [PC(34:2), plasmalogen PC(36:4)/PC(O-36:5), and a compound with m/z of 781.0545], two CpGs (on the DHCR24 and SC4MOL gene), and two proteins (periostin and CCL22). CCL22, a macrophage-derived chemokine has not been previously identified in relation to birthweight. Since the results of the omics integration indicated the central role of cholesterol metabolism, we explored the association of cholesterol levels in cord blood with birthweight in the ENVIRONAGE cohort (n = 1097), finding that higher birthweight was associated with increased high-density lipoprotein cholesterol and that high-density lipoprotein cholesterol was lower in small versus large for gestational age newborns. Conclusions Our data suggests that an integration of different omic-layers in addition to single omics studies is a useful approach to generate new hypotheses regarding biological mechanisms. CCL22 and cholesterol metabolism in cord blood play a mechanistic role in birthweight., Highlights • Using multiple omics, we provide an unprecedented window into the biological processes underlying birthweight. • We identified molecular signals never previously linked to birthweight, e.g. gene expression of JAK3 and chemokine CCL22. • Our data suggested that cholesterol and related metabolic pathways are related to birthweight. • The identified signals may create a molecular basis for the onset of health outcomes associated with birthweight variation.

Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis., Graphical abstract Illustration of direct protein S-nitrosylation (SNO) in protein folding and degradation pathways. Key SNO-targets identified and studied in the present study are HSPA8, and UBE2D isoenzymes. SNOing of Cys17 of HSPA8 likely compromises binding of ATP/ADP, which is essential for HSPA8's functions including protein folding, clathrin uncoating, protein shuttling to and from organelles, chaperone-mediated-autophagy (CMA) and chaperone assisted autophagy (CASA) and proteasomal degradation of specific proteins such as beta-actin. SNOing of UBE2D's catalytic site cysteine reduces its activity and interferes with the degradation of specific proteins, which rely on ubiquitination via UBE2D such as p53. Abbreviations, CMA, Chaperone mediated autophagy; CASA, Chaperone assisted autophagy; ERAD, ER associated degradation; UPS, Ubiquitin-Proteasome System; SASP, Senescence associated secretory phenotype; UPR, unfolded protein response; NOS, nitric oxide synthase; BH4, tetrahydrobiopterinfx1, Highlights • Direct NO-mediated S-nitrosylation of HSAP8 interferes with chaperone-mediated autophagy. neuroblastoma cells. • Direct S-nitrosylation of UBE2D reduces ubiquitination and proteasomal degradation proteins. • NO-evoked disturbances of protein homeostasis lead to an aged and senescent phenotype. • NO-evoked pro-aging effects agree with NOS upregulations in the aging rodent brain.

Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets., Graphical abstract, Highlights • Adaptation of a fully defined, holidic food source for fruit fly SILAC (SILAF) • More than 99% labeling efficiency with lysine-6 within one larval generation • One milligram of fully lysine-6 labeled peptides for less than $15 • 14,000 Drosophila phosphorylated sites with 1500 occupancy values • Mitochondrial DmLRPPRC1 loss increases phosphorylation of NDUFB10 and NDUFA4, In Brief We advanced a fully composed food source for Drosophila melanogaster into a highly efficient, versatile, and cheap labeling method, termed SILAF. The larval proteome incorporates more than 99% heavy lysine-6 label within 6 days, while the adult fly metabolism allows protein turnover studies. We obtained a phosphoproteome with site-specific occupancy in a fly model of mitochondrial metabolic disease, highlighting the regulation of two novel conserved phosphosites on subunits of the electron transport chain.