TFEB-dependent lysosome biogenesis is required for senescence.

The accumulation of senescent cells is recognised as a driver of tissue and organismal ageing. One of the gold-standard hallmarks of a senescent cell is an increase in lysosomal content, as measured by senescence-associated ß-galactosidase (Senß-Gal) activity. The lysosome plays a central role in integrating mitogenic and stress cues to control cell metabolism, which is known to be dysregulated in senescence. Despite this, little is known about the cause and consequence of lysosomal biogenesis in senescence. We find here that lysosomes in senescent cells are dysfunctional; they have higher pH, increased evidence of membrane damage and reduced proteolytic capacity. The significant increase in lysosomal content is however sufficient to maintain degradative capacity of the cell to a level comparable to proliferating control cells. We demonstrate that increased nuclear TFEB/TFE3 supports lysosome biogenesis, is a hallmark of multiple forms of senescence and is required for senescent cell survival. TFEB/TFE3 are hypo-phosphorylated and show constitutive nuclear localisation in senescence. Evidence suggests that several pathways may contribute to TFEB/TFE3 dysregulation in senescence.

Recovery of Water Homeostasis in Adenine-Induced Kidney Disease Is Mediated by Increased AQP2 Membrane Targeting.

Chronic kidney disease (CKD) represents a major public health burden with increasing prevalence. Current therapies focus on delaying CKD progression, underscoring the need for innovative treatments. This necessitates animal models that accurately reflect human kidney pathologies, particularly for studying potential reversibility and regenerative mechanisms, which are often hindered by the progressive and irreversible nature of most CKD models. In this study, CKD was induced in mice using a 0.2% adenine-enriched diet for 4 weeks, followed by a recovery period of 1 or 2 weeks. The aim was to characterize the impact of adenine feeding on kidney function and injury as well as water and salt homeostasis throughout disease progression and recovery. The adenine diet induced CKD is characterized by impaired renal function, tubular injury, inflammation, and fibrosis. A significant decrease in urine osmolality, coupled with diminished aquaporin-2 (AQP2) expression and membrane targeting, was observed after adenine treatment. Intriguingly, these parameters exhibited a substantial increase after a two-week recovery period. Despite these functional improvements, only partial reversal of inflammation, tubular damage, and fibrosis were observed after the recovery period, indicating that the inclusion of the molecular and structural parameters is needed for a more complete monitoring of kidney status.

oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins.

Site-selective disulfide rebridging has emerged as a powerful strategy to modulate the structural and functional properties of proteins. Here, we introduce a novel class of electrophilic reagents, designated oxSTEF, that demonstrate excellent efficiency in disulfide rebridging via double thiol exchange. The oxSTEF reagents are prepared using an efficient synthetic sequence which may be diverted to obtain a range of derivatives allowing for tuning of reactivity or steric bulk. We demonstrate highly selective rebridging of cyclic peptides and native proteins, such as human growth hormone, and the absence of cross-reactivity with other nucleophilic amino acid residues. The oxSTEF conjugates undergo glutathione-mediated disintegration under tumor-relevant glutathione concentrations, which highlights their potential for use in targeted drug delivery. Finally, the α-dicarbonyl motif of the oxSTEF reagents enables "second phase" oxime ligation, which furthermore increases the thiol stability of the conjugates significantly.

Extracellular vesicles of the probiotic bacteria E. coli O83 activate innate immunity and prevent allergy in mice.

BACKGROUND: E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. METHODS: EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. CONCLUSIONS: We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Video Abstract.

Differences in the urinary metabolome and proteome between wet and dry nights in children with monosymptomatic nocturnal enuresis and nocturnal polyuria.

BACKGROUND: Nocturnal enuresis (NE) is a common disease with multiple pathogenic mechanisms. This study aimed to compare levels of metabolites and proteins between wet and dry nights in urine samples from children with monosymptomatic NE (MNE). METHODS: Ten boys with MNE and nocturnal polyuria (age: 7.6 ± 1.3 years) collected their total nighttime urine production during a wet and a dry night. Untargeted metabolomics and proteomics were performed on the urine samples by liquid chromatography coupled with high-mass accuracy tandem mass spectrometry (LC-MS/MS). RESULTS: On wet nights, we found reduced urine osmolality (P = 0.025) and increased excretion of urinary potassium and sodium by a factor of, respectively, 2.1 (P = 0.038) and 1.9 (P = 0.19) compared with dry nights. LC-MS identified 59 metabolites and 84 proteins with significantly different levels between wet and dry nights (fold change (FC) < 0.67 or > 1.5, P < 0.05). Some compounds were validated by different methodologies. During wet nights, levels of compounds related to oxidative stress and blood pressure, including adrenalin, were increased. We found reduced levels of aquaporin-2 on wet nights. The FCs in the 59 metabolites were positively correlated to the FCs in the same metabolites identified in urine samples obtained during the evening preceding wet and dry nights. CONCLUSIONS: Oxidative stress, which in the literature has been associated with nocturia and disturbances in sleep, might be increased during wet nights in children with MNE. We further found evidence of increased sympathetic activity. The mechanisms related to having wet nights in children with MNE seem complex, and both free water and solute handling appear to be important. A higher resolution version of the Graphical abstract is available as Supplementary information.

α-Lactam Electrophiles for Covalent Chemical Biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3-membered N-heterocyclic compounds-such as aziridines, azirines, and oxaziridines-possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

Automated Solid-Phase Oligo(disulfide) Synthesis.

A method for automated solid-phase synthesis of oligo(disulfide)s was developed. It is based on a synthetic cycle comprising removal of a protecting group from a resin-bound thiol followed by treatment with monomers containing a thiosulfonate as an activated precursor. For ease of purification and characterization, the disulfide oligomers were synthesized as extensions of oligonucleotides on an automated oligonucleotide synthesizer. Six different dithiol monomer building blocks were synthesized. Sequence-defined oligomers of up to seven disulfide units were synthesized and purified. The sequence of the oligomer was confirmed by tandem MS/MS analysis. One of the monomers contains a coumarin cargo that can be released by a thiol-mediated release mechanism. When the monomer was incorporated into an oligo(disulfide) and subjected to reducing conditions, the cargo was released under near-physiological conditions, which underlines the potential use of these molecules in drug delivery systems.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

Aquaporin-5 regulation of cell-cell adhesion proteins: an elusive "tail" story.

Aquaporins (AQPs) are water channels that facilitate transport of water across cellular membranes. AQPs are overexpressed in several cancers. Especially in breast cancer, AQP5 overexpression correlates with spread to lymph nodes and poor prognosis. Previously, we showed that AQP5 expression reduced cell-cell adhesion by reducing levels of adherens and tight-junction proteins (e.g., ZO-1, plakoglobin, and ß-catenin) at the actual junctions. Here, we show that, when targeted to the plasma membrane, the AQP5 COOH-terminal tail domain regulated junctional proteins and, moreover, that AQP5 interacted with ZO-1, plakoglobin, ß-catenin, and desmoglein-2, which were all reduced at junctions upon AQP5 overexpression. Thus, our data suggest that AQP5 mediates the effect on cell-cell adhesion via interactions with junctional proteins independently of AQP5-mediated water transport. AQP5 overexpression in cancers may thus contribute to carcinogenesis and cancer spread by two independent mechanisms: reduced cell-cell adhesion, a characteristic of epithelial-mesenchymal transition, and increased cell migration capacity via water transport.

A Reagent for Amine-Directed Conjugation to IgG1 Antibodies.

Functionalized antibodies are an indispensable resource for diagnosis, therapy and as a research tool for chemical biology. However, simpler and better methodologies are often required to improve the labeling of antibodies in terms of selectivity and scalability. Herein, we report the development of an easily available chemical reagent that allows site-directed labeling of native human IgG1 antibodies in good yield and mono-labeling selectivity. The salicylaldehyde moiety of the reagent reacts with surface exposed lysine residues to transiently form an iminium ion, and this positions a semi-reactive ester in proximity of a second lysine residue that reacts with the ester to form an amide. Interestingly, it appears that the formation of the iminium ion also has a significant activating effect of the ester. We use flow cytometry and bio-layer interferometry to confirm that the labeled antibodies retain antigen binding.

Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.

Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid ß-oxidation (FAO), and the tricarboxylic acid cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.

Introduction of an Aldehyde Handle on Nanobodies by Affinity-Guided Labeling.

Chemically modified antigen-binding proteins are widely applied for their targeting abilities in the fields of biotechnology, medicine, and diagnostics. However, the production of site-selectively modified proteins remains a challenge. Here, we have designed a chemical probe for the introduction of a reactive aldehyde on nanobodies by metal-complex-guided conjugation. The probe design allows for purification of the conjugates, and the aldehyde constitutes an efficient handle for further modification of the nanobodies. In vitro experiments confirmed the binding activity and selectivity of fluorescent conjugates toward the native antigen. Furthermore, the modification strategy allowed for production of a nanobody-drug conjugate that was active in vitro.

Brain proteome changes in female Brd1+/- mice unmask dendritic spine pathology and show enrichment for schizophrenia risk.

Genetic and molecular studies have implicated the Bromodomain containing 1 (BRD1) gene in the pathogenesis of schizophrenia and bipolar disorder. Accordingly, mice heterozygous for a targeted deletion of Brd1 (Brd1+/- mice) show behavioral phenotypes with broad translational relevance to psychiatric disorders. BRD1 encodes a scaffold protein that affects the expression of many genes through modulation of histone acetylation. BRD1 target genes have been identified in cell lines; however the impact of reduced Brd1 levels on the brain proteome is largely unknown. In this study, we applied label-based quantitative mass spectrometry to profile the frontal cortex, hippocampus and striatum proteome and synaptosomal proteome of female Brd1+/- mice. We successfully quantified between 1537 and 2196 proteins and show widespread changes in protein abundancies and compartmentalization. By integrative analysis of human genetic data, we find that the differentially abundant proteins in frontal cortex and hippocampus are enriched for schizophrenia risk further linking the actions of BRD1 to psychiatric disorders. Affected proteins were further enriched for proteins involved in processes known to influence neuronal and dendritic spine morphology e.g. regulation of cytoskeleton dynamics and mitochondrial function. Directly prompted in these findings, we investigated dendritic spine morphology of pyramidal neurons in anterior cingulate cortex and found them significantly altered, including reduced size of small dendritic spines and decreased number of the mature mushroom type. Collectively, our study describes known as well as new mechanisms related to BRD1 dysfunction and its role in psychiatric disorders, and provides evidence for the molecular and cellular dysfunctions underlying altered neurosignalling and cognition in Brd1+/- mice.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Aptamer-Directed Conjugation of DNA to Therapeutic Antibodies.

A method for aptamer directed conjugation of DNA to therapeutic antibodies has been developed. In the method, an antibody selective aptamer binds to a specific site in the constant domain of human IgG1 antibodies and is used for both templated and direct conjugation to the antibodies. Through optimization of the design and reaction conditions, the antibody-DNA conjugates could be produced efficiently using equal stoichiometry of protein and DNA. Three different antibodies were evaluated, and the conjugates were characterized by anion exchange chromatography and SDS-PAGE. The conjugation sites for one of the antibodies were determined by MS/MS analysis of the digested conjugate. The antibody-DNA conjugate was also tested for receptor binding on cell surfaces showing retained binding.

Formation of i-motifs from acyclic (l)-threoninol nucleic acids.

Acyclic (l)-threoninol nucleic acids ((l)-aTNA) containing poly-cytosines are prepared and investigated at various pH values, revealing the formation of a highly stable structure at lower pH that have the characteristics of an i-motif. Depending on the sequence, the aTNA forms inter-, bi- and intra-molecular i-motif structures. Pyrene was conjugated to aTNA sequences and both monomeric and excimer fluorescence were efficiently quenched by the i-motif structures and thus demonstrated that the aTNA i-motif can serve as a pH switch.

Imidazole carbamate probes for affinity guided azide-transfer to metal-binding proteins.

Probes for affinity guided conjugation have shown great promise for the preparation of high-quality protein conjugates. However, such probes are often limited in their protein scope. Here, we demonstrate the synthesis and use of imidazole carbamate probes for affinity guided conjugation of an azide handle to a wide variety of metal binding proteins such as antibodies, enzymes, nanobodies and His6-tagged proteins. The azide handles were used to link dyes to the proteins for gel analysis and binding studies. In a comparative study we find that the probe containing three NTA groups show higher selectivity than the probe containing two. In a mixture of proteins, the probe shows preference for labelling His6-tagged proteins.

A Cyclopropene Electrophile that Targets Glutathione S-Transferase Omega-1 in Cells.

Cyclopropenes are an important new addition to the portfolio of functional groups that can be used for bioorthogonal couplings. The inert nature of these highly strained compounds in complex biological systems is almost counterintuitive given their established electrophilic properties in organic synthesis. Here we provide the first demonstration of a cyclopropene that is capable of direct conjugation to protein targets in cells and show that this compound preferentially alkylates the active site cysteine of glutathione S-transferase omega-1 (GSTO1).

STEFs: Activated Vinylogous Protein-Reactive Electrophiles.

Reported here is the synthesis of a class of semi-oxamide vinylogous thioesters, designated STEFs, and the use of these agents as new electrophilic warheads. This work includes preparation of simple probes that contain this reactive motif as well as its installation on a more complex kinase inhibitor scaffold. A key aspect of STEFs is their reactivity towards both thiol and amine groups. Shown here is that amine conjugations in peptidic and proteinogenic samples can be facilitated by initial, fast conjugation to proximal thiol residues. Evidence that both the selectivity and the reactivity can be tuned by the structure of STEFs is provided, and given the unique ability of this functionality to conjugate by an addition-elimination mechanism, STEFs are electrophilic warheads that could find broad use in chemical biology.

Proteomics of the Rat Myocardium during Development of Type 2 Diabetes Mellitus Reveals Progressive Alterations in Major Metabolic Pathways.

Congestive heart failure and poor clinical outcome after myocardial infarction are known complications in patients with type-2 diabetes mellitus (T2DM). Protein alterations may be involved in the mechanisms underlying these disarrays in the diabetic heart. Here we map proteins involved in intracellular metabolic pathways in the Zucker diabetic fatty rat heart as T2DM develops using MS based proteomics. The prediabetic state only induced minor pathway changes, whereas onset and late T2DM caused pronounced perturbations. Two actin-associated proteins, ARPC2 and TPM3, were up-regulated at the prediabetic state indicating increased actin dynamics. All differentially regulated proteins involved in fatty acid metabolism, both peroxisomal and mitochondrial, were up-regulated at late T2DM, whereas enzymes of branched chain amino acid degradation were all down-regulated. At both onset and late T2DM, two members of the serine protease inhibitor superfamily, SERPINA3K and SERPINA3L, were down-regulated. Furthermore, we found alterations in proteins involved in clearance of advanced glycation end-products and lipotoxicity, DCXR and CBR1, at both onset and late T2DM. These proteins deserve elucidation with regard to their role in T2DM pathogenesis and their respective role in the deterioration of the diabetic heart. Data are available via ProteomeXchange with identifiers PXD009538, PXD009554, and PXD009555.