Biology subjects

Stein, F.

Publications and source records attributed to Stein, F..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity

IntroductionAssessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. ObjectivesIn a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. MethodsMonoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. ResultsChemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. ConclusionOur ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.

plant biology↗

bioRxiv2023

Connectome architecture shapes large-scale cortical reorganization in schizophrenia: a worldwide ENIGMA study

While schizophrenia is considered a prototypical network disorder characterized by widespread brain-morphological alterations, it still remains unclear whether distributed structural alterations robustly reflect underlying network layout. Here, we tested whether large-scale structural alterations in schizophrenia relate to normative structural and functional connectome architecture, and systematically evaluated robustness and generalizability of these network-level alterations. Leveraging anatomical MRI scans from 2,439 adults with schizophrenia and 2,867 healthy controls from 26 ENIGMA sites and normative data from the Human Connectome Project (n=207), we evaluated structural alterations of schizophrenia against two network susceptibility models: i) hub vulnerability, which examines associations between regional network centrality and magnitude of disease-related alterations; ii) epicenter mapping, which identify regions whose typical connectivity profile most closely resembles the disease-related morphological alterations. To assess generalizability and specificity, we contextualized the influence of site, disease stages, and individual clinical factors and compared network associations of schizophrenia with that found in affective disorders. Schizophrenia-related structural alterations co-localized with interconnected functional and structural hubs and harbored temporo-paralimbic and frontal epicenters. Findings were robust across sites and related to individual symptom profiles. We observed localized unique epicenters for first-episode psychosis and early stages, and transmodal epicenters that were shared across first-episode to chronic stages. Moreover, transdiagnostic comparisons revealed overlapping epicenters in schizophrenia and bipolar, but not major depressive disorder, yielding insights in pathophysiological continuity within the schizophrenia-bipolar-spectrum. In sum, cortical alterations over the course of schizophrenia robustly follow brain network architecture, emphasizing marked hub susceptibility and temporo-frontal epicenters at both the level of the group and the individual. Subtle variations of epicenters across disease stages suggest interacting pathological processes, while associations with patient-specific symptoms support additional inter-individual variability of hub vulnerability and epicenters in schizophrenia. Our work contributes to recognizing potentially common pathways to better understand macroscale structural alterations, and inter-individual variability in schizophrenia.

neuroscience↗

bioRxiv2022

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger the mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling (ppTPP). This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labeling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Strikingly, different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of the functional modules that are relevant for mitoprotein-induced stress. Thus, our new ppTPP approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of protein abundance and protein stability.

biochemistry↗

bioRxiv2022

A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organellar membranes from yeast, MemPrep. We demonstrate the purity of our ER preparations by quantitative proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of the combined preparative and analytical platform to acutely stressed cells reveals dynamic ER membrane remodeling and establishes molecular fingerprints of lipid bilayer stress.

cell biology↗

bioRxiv2022

Transient inhibition of translation improves long-term cardiac function after ischemia/reperfusion by attenuating the inflammatory response

RationaleRapid reperfusion is the most effective treatment for attenuating cardiac injury caused by myocardial ischemia. Yet, reperfusion itself elicits damage to the myocardium through incompletely understood mechanisms, known as ischemia/reperfusion (I/R) injury. The myocardium adapts to I/R by changes in gene expression, which determines the cellular response to reperfusion. Protein translation is a key component of gene expression. However, it is unknown how regulation of translation contributes to cardiac gene expression in response to reperfusion and whether it can be targeted to mitigate I/R injury. MethodsTo examine translation and its impact on gene expression in response to I/R we assessed protein synthesis at different timepoints after ischemia and reperfusion in vitro and in vivo. Pharmacological inhibitors were used to dissect the underlying molecular mechanisms of translational control. Transient inhibition of protein synthesis was undertaken to decipher the effects of the translational response to reperfusion on cardiac function and inflammation. Cell-type-specific ribosome profiling was performed in mice subjected to I/R to determine the impact of translation on the regulation of gene expression in cardiomyocytes. ResultsReperfusion increased translation rates from a previously suppressed state during ischemia in cardiomyocytes, which was associated with the induction of cell death. In vivo, I/R resulted in strong activation of translation in the myocardial border zone. Detailed analysis revealed that the upregulation of translation is mediated by eIF4F complex formation, which was specifically mediated by the mTORC1-4EBP1-eIF4F axis. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin, respectively, attenuated translation, reduced infarct size and improved long-term cardiac function after myocardial infarction. Cardiomyocyte-specific ribosome profiling identified that reperfusion damage increased translation of mRNA networks in cardiomyocytes associated with cardiac inflammation and cell infiltration. Transient inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory transcripts such as Ccl2, thereby reducing Ly6Chi monocyte infiltration and myocardial inflammation. ConclusionsMyocardial reperfusion induces protein synthesis in the border zone which contributes to I/R injury by rapidly translating a specific maladaptive mRNA network that mediates immune cell infiltration and inflammation. Transient inhibition of the mTORC1-4EBP1-eIF4F signaling axis during reperfusion attenuates this proinflammatory translational response, protects against I/R injury and improves long-term cardiac function after myocardial infarction. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis is the first study to investigate the impact of translational regulation on cardiomyocyte gene expression in response to myocardial ischemia/reperfusion. C_LIO_LIWe show that translation regulates approximately two-thirds of differentially expressed genes in cardiomyocytes after ischemia/reperfusion, including many involved in inflammation and immune cell infiltration. C_LIO_LIThe translational response to ischemia/reperfusion is regulated by the mTORC1-4EBP1-eIF4F axis, which determines pro-inflammatory monocyte infiltration via control of the expression of the chemokine Ccl2. C_LI What Are the Clinical Implications?O_LICurrently, there are no specific therapies to prevent ischemia/reperfusion injury, which is mediated, at least in part, by a maladaptive inflammatory response. C_LIO_LIA translationally controlled network regulated by the mTORC1-4EBP1-eIF4F axis can be targeted by a short-term pharmacological intervention to attenuate the inflammatory response and improve cardiac function after ischemia/reperfusion in mice. C_LIO_LIThis study supports the emerging concept of selectively inhibiting maladaptive elements of the inflammatory response to improve outcome in patients after myocardial infarction; in addition, it provides a mechanistic basis for the currently ongoing CLEVER-ACS trial. C_LI

molecular biology↗

bioRxiv2022

Interrelated effects of age and parenthood on whole-brain controllability: protective effects of parenthood in mothers

BackgroundControllability is a measure of the brains ability to orchestrate neural activity which can be quantified in terms of properties of the brains network connectivity. Evidence from the literature suggests that aging can exert a general effect on whole-brain controllability. Mounting evidence, on the other hand, suggests that parenthood and motherhood in particular lead to long-lasting changes in brain architecture that effectively slow down brain aging. We hypothesize that parenthood might preserve brain controllability properties from aging. MethodsIn a sample of 814 healthy individuals (aged 33.9{+/-}12.7 years, 522 females), we estimate whole-brain controllability and compare the aging effects in subjects with vs. those without children. We use diffusion tensor imaging (DTI) to estimate the brain structural connectome. The level of brain control is then calculated from the connectomic properties of the brain structure. Specifically, we measure the network control over many low-energy state transitions (average controllability) and the network control over difficult-to-reach states (modal controllability). Results and conclusionIn nulliparous females, whole-brain average controllability increases, and modal controllability decreases with age, a trend that we do not observe in parous females. Statistical comparison of the controllability metrics shows that modal controllability is higher and average controllability is lower in parous females compared to nulliparous females. In men, we observed the same trend, but the difference between nulliparous and parous males do not reach statistical significance. Our results provide strong evidence that parenthood contradicts aging effects on brain controllability and the effect is stronger in mothers.

neuroscience↗

bioRxiv2022

The RNA-binding protein landscapes differ between mammalian organs and cultured cells

System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo eRIC (enhanced RNA interactome capture) to characterize the poly(A)RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

molecular biology↗

bioRxiv2021

Lysosome-targeted lipid probes reveal sterol transporters NPC1 and LIMP2 as sphingosine transporters

Lysosomes are central catabolic organelles involved in lipid homeostasis and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions as well as their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photo-crosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and LIMP-2/SCARB2, also directly bind to sphingosine. In addition, we showed that absence of either protein leads to lysosomal sphingosine accumulation which suggests a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

cell biology↗

bioRxiv2021

Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 L-type voltage gated calcium channel subunit CACNB2

Bipolar disorder (BD) is a chronic mood disorder characterized by alternating manic and depressive episodes, often in conjunction with cognitive deficits. Dysregulation of neuroplasticity and calcium homeostasis as a result of complex genetic environment interactions are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that a BD-associated microRNA, miR-499-5p, regulates neuronal dendrite development and cognitive function by downregulating the BD risk gene CACNB2. miR-499-5p expression is increased in peripheral blood of BD patients and healthy subjects at risk of developing the disorder due to a history of childhood maltreatment. This up-regulation is paralleled in the hippocampus of rats which underwent juvenile social isolation. Elevating miR-499-5p levels in rat hippocampal pyramidal neurons impairs dendritogenesis and reduces surface expression and activity of the voltage-gated L-type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory {beta}-subunit of Cav1.2, as a direct target of miR-499-5p in neurons. CACNB2 downregulation is required for the miR-499-5p dependent impairment of dendritogenesis, suggesting that CACNB2 is an important downstream target of miR-499-5p in the regulation of neuroplasticity. Finally, elevating miR-499-5p in the hippocampus in vivo is sufficient to induce short-term memory impairments in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. Taken together, we propose that stress-induced upregulation of miR-499-5p contributes to dendritic impairments and deregulated calcium homeostasis in BD, with specific implications for the neurocognitive dysfunction frequently observed in BD patients.

neuroscience↗

bioRxiv2021

Increased levels of the mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient material and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but the causalities remained unclear. We used yeast as model system to analyze the relevance of mitochondrial processes for the behavior of an aggregation-prone polyQ protein derived from human huntingtin. Induction of Q97-GFP rapidly leads to insoluble cytosolic aggregates and cell death. Although this aggregation impairs mitochondrial respiration only slightly, it interferes with efficient import of mitochondrial precursor proteins. Mutants in the import component Mia40 are hypersensitive to Q97-GFP. Even more surprisingly, Mia40 overexpression strongly suppresses the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the posttranslational import into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Owing to its rate-limiting role for mitochondrial protein import, Mia40 acts as a regulatory component in this competition. This role of Mia40 as dynamic regulator in mitochondrial biogenesis can apparently be exploited to stabilize cytosolic proteostasis. (174/175 words)

biochemistry↗

bioRxiv2020

Pharmacological inhibition of nonsense-mediated RNA decay augments HLA class I-mediated presentation of neoepitopes in MSI CRC

Microsatellite-unstable (MSI) colorectal cancer is characterized by the accumulation of somatic insertion/deletion (InDel) mutations potentially generating tumor-specific, frameshifted protein sequences. Such mutations typically generate premature translation termination codons targeting the affected mRNAs to degradation by nonsense-mediated RNA decay (NMD), limiting the synthesis and HLA class I-mediated presentation of tumor-specific InDel neoepitopes. We reasoned that the NMD inhibitor 5-azacytidine (5AZA) could serve to increase the expression of NMD-sensitive neoepitopes and analyzed the immunopeptidome of MSI HCT-116 cells using a proteogenomic approach. After immunoprecipitation of HLA:peptide complexes, we identified more than 10,000 HLA class I-presented peptides by LC-MS/MS including five InDel neoepitopes. The InDel neoepitopes were verified on the genomic, transcriptomic, and peptidomic level. Treatment with 5AZA increased the expression of the corresponding frameshift- and premature termination codon-bearing mRNAs and enhanced the presentation of peptides originating from known NMD targets and one of the identified InDel neoepitopes. By analyzing an array of MSI colorectal cancer cell lines and patient samples, we found the underlying frameshift mutation to be highly recurrent and immunization with the corresponding neoepitope induced strong CD8+ T cell responses in an HLA-A*02:01 transgenic mouse model. Our data directly show that peptides originating from frameshifted open reading frames due to InDel mutations in mismatch repair-deficient cells are presented on the cell surface via HLA class I. Moreover, we demonstrate the utility of NMD inhibitor-enhanced HLA class I-mediated presentation of InDel neoepitopes as well as their immunogenicity, uncovering the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies. One Sentence SummaryImmunopeptidomics identified increased HLA class I-mediated presentation of immunogenic, frameshift-derived neoepitopes following NMD inhibition.

cancer biology↗

bioRxiv2020

Bacterial retrons encode tripartite toxin/antitoxin systems

Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate. Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems.

microbiology↗

bioRxiv2020

Global mapping of Salmonella enterica-host protein-protein interactions during infection

Intracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to the Salmonella Containing Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

microbiology↗

bioRxiv2020

Stroma transcriptomic and proteomic profile of prostate cancer metastasis xenograft models reveals conservation of bonemicroenvironment signatures

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men with therapy resistance acquisition to androgen deprivation treatment and metastasis progression. Understanding the mechanisms of tumor progression to metastatic stage is necessary for the design of therapeutic and prognostic schemes. The main objective of the current study is to determine, using transcriptomic and proteomic analyses on patient derived-xenograft models, whether differentially aggressive PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern, and how this information could be applied in prognostics. Transcriptomic profiling (RNA Sequencing) was performed on PCa PDX models representing different disease stages; BM18 (androgen dependent bone metastasis) and LAPC9 (androgen independent bone metastasis). Using organism-specific reference databases, the human-specific transcriptome, representing the tumor, was identified and separated from the mouse-specific transcriptome (representing the contributing stroma counterpart) from the same PDX tumor samples. To identify proteome changes in the tumor (human) versus the stroma (mouse), we performed human and mouse cell separation using the MACS mouse depletion sorting kit, and subjected protein lysates to quantitative TMT labeling and mass spectrometry. We show that tenascin C is one of the most abundant stromal genes in bone metastasis PCa PDXs, is modulated by androgen levels in vivo and is highly expressed in castration resistant LAPC9 PDX compared to castration sensitive BM18 PDX. Tissue microarray of primary PCa samples (N=210) was used to evaluate the potential of TNC to act as a metastasis prognosis marker. Low number of TNC-positive cells were associated with statistically significant clinical progression to local recurrence or metastasis, compared to high TNC-positive group. Our data showed that metastatic PCa PDXs that differ in androgen sensitivity trigger a differential stroma response suggesting that stroma was influenced by tumor cues. Selected stromal markers of osteoblastic PCa induced bone metastases, were induced in the microenvironment of the host organism in metastatic xenografts, although implanted in a non-bone site, indicating a conserved mechanism of tumor cells to induce a stromal pre-metastatic signature with high potential prognostic or diagnostic value.

cancer biology↗

bioRxiv2020

Long-term synaptic depression triggers local biogenesis of autophagic vesicles in dendrites and requires autophagic degradation

In neurons, biogenesis of autophagic vesicles (AVs) is spatially confined to the axon tip under baseline conditions. However, it remains unknown whether their biogenesis can be induced in other neuronal compartments following synaptic activity in order to serve local functions. Here, we show that both major types of long-term synaptic depression (LTD), a form of plasticity expressed by the shrinkage and elimination of dendritic spines, trigger the rapid and local biogenesis of AVs in post-synaptic dendrites. In return, autophagy is indispensable for LTD, as either genetic ablation of atg5 in pyramidal neurons or acute pharmacological inhibition of AV biogenesis totally prevents LTD induction. Using quantitative proteomic profiling of purified AVs, we reveal that upon LTD the autophagic cargo is significantly enriched for synaptic proteins, as well as modulators of the actin cytoskeleton and autism-implicated proteins. In line with these findings, a mild autophagy deficit is sufficient to impair behavioral flexibility, a cognitive function that requires efficient LTD. Therefore, local synthesis and assembly of the autophagic machinery in dendrites ensure the elimination of synaptic structures via degradation of their components, facilitating plasticity and associated behaviors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/983965v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1a7bc14org.highwire.dtl.DTLVardef@12cece0org.highwire.dtl.DTLVardef@738687org.highwire.dtl.DTLVardef@8518af_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefKallergi, Daskalaki and colleagues demonstrate that autophagy is cell autonomously required in pyramidal excitatory neurons for the induction of long-term synaptic depression (LTD). They uncover the novel and local biogenesis of autophagic vesicles (AVs) in dendrites upon LTD, by which post-synaptic components are rapidly accessible on-site for autophagic degradation. Using quantitative proteomics on purified AVs, they reveal that upon LTD the autophagic cargo is enriched in synaptic, cytoskeletal and autism-implicated proteins. HighlightsO_LIAutophagy is required cell-autonomously in pyramidal neurons for LTD. C_LIO_LINMDAR- and mGluR-mediated LTD trigger the local biogenesis of autophagic vesicles in dendrites. C_LIO_LIAutophagic vesicles sequester primarily synaptic and cytoskeletal cargo upon LTD. C_LIO_LIMild impairment in autophagy leads to deficits in cognitive flexibility. C_LI

neuroscience↗

bioRxiv2020

Aggregation and Disaggregation Features of the Human Proteome

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry-based proteomics to characterize aggregation and disaggregation in human cells after non-lethal heat shock. Aggregation-prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions also resulted in faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared to control condition. Our results provide a rich resource of heat stress-related protein solubility data, propose novel roles for intrinsically disordered regions in protein quality control and reveal a protection mechanism to repress protein aggregation in heat stress.

systems biology↗

bioRxiv2019

Multiomic and quantitative label-free microscopy-based analysis of ex vivo culture and TGFbeta1 stimulation of human precision-cut lung slices

Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant hurdle for discovering cures for fibrosis is the lack of suitable models and techniques to quantify mature collagen deposition in tissues. Here we have extensively characterized an ex-vivo cultured human lung derived, precision-cut lung slices model (hPCLS) using live fluorescence light microscopy as well as mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint. Using an integrated approach of multiple readouts such as quantitative label-free Second Harmonic Generation (SHG) imaging to measure fibrillar collagen in the extracellular matrix and ELISA-based methods to measure soluble ECM biomarkers, we investigated TGFbeta1-mediated pro-fibrotic signalling in hPCLS. We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cells surviving for at least two weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed strong induction of ECM synthesis proteins P1NP and fibronectin upon TGFb stimulation. Importantly, this effect translated into an increased deposition of fibrillar collagen in ECM of cultured hPCLS as measured by a novel quantitative SHG-based imaging method only following addition of a metalloproteinase inhibitor (GM6001). Together the data show that an integrated approach of measuring soluble pro-fibrotic markers and quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.

physiology↗

bioRxiv2019

Towards a systematic map of the functional role of protein phosphorylation

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphorylation sites have a known function. Here, we devised a chemical genetic approach to study the functional relevance of phosphorylation in S. cerevisiae. We generated 474 phospho-deficient mutants that, along with the gene deletion library, were screened for fitness in 102 conditions. Of these, 42% exhibited growth phenotypes, suggesting these phosphosites are likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. While some phosphomutants showed loss-of-function phenotypes, a higher fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This study provides a roadmap for functionally characterizing phosphorylation in a systematic manner.

systems biology↗