Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Proteomic analysis of the urothelial cancer landscape.

Urothelial bladder cancer (UC) has a wide tumor biological spectrum with challenging prognostic stratification and relevant therapy-associated morbidity. Most molecular classifications relate only indirectly to the therapeutically relevant protein level. We improve the pre-analytics of clinical samples for proteome analyses and characterize a cohort of 434 samples with 242 tumors and 192 paired normal mucosae covering the full range of UC. We evaluate sample-wise tumor specificity and rank biomarkers by target relevance. We identify robust proteomic subtypes with prognostic information independent from histopathological groups. In silico drug prediction suggests efficacy of several compounds hitherto not in clinical use. Both in silico and in vitro data indicate predictive value of the proteomic clusters for these drugs. We underline that proteomics is relevant for personalized oncology and provide abundance and tumor specificity data for a large part of the UC proteome ( www.cancerproteins.org ).

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis.

BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the "metastasis-suppressor" effect of HERC5, with a focus on mitochondrial metabolism pathways. METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

16S rRNA sequencing analysis of the oral and fecal microbiota in colorectal cancer positives versus colorectal cancer negatives in Iranian population.

BACKGROUND: Colorectal cancer (CRC) poses a significant healthcare challenge, accounting for nearly 6.1% of global cancer cases. Early detection, facilitated by population screening utilizing innovative biomarkers, is pivotal for mitigating CRC incidence. This study aims to scrutinize the fecal and salivary microbiomes of CRC-positive individuals (CPs) in comparison to CRC-negative counterparts (CNs) to enhance early CRC diagnosis through microbial biomarkers. MATERIAL AND METHODS: A total of 80 oral and stool samples were collected from Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, encompassing both CPs and CNs undergoing screening. Microbial profiling was conducted using 16S rRNA sequencing assays, employing the Nextera XT Index Kit on an Illumina NovaSeq platform. RESULTS: Distinct microbial profiles were observed in saliva and stool samples of CPs, diverging significantly from those of CNs at various taxonomic levels, including phylum, family, and species. Saliva samples from CPs exhibited abundance of Calothrix parietina, Granulicatella adiacens, Rothia dentocariosa, and Rothia mucilaginosa, absent in CNs. Additionally, Lachnospiraceae and Prevotellaceae were markedly higher in CPs' feces, while the Fusobacteria phylum was significantly elevated in CPs' saliva. Conversely, the non-pathogenic bacterium Akkermansia muciniphila exhibited a significant decrease in CPs' fecal samples compared to CNs. CONCLUSION: Through meticulous selection of saliva and stool microbes based on Mean Decrease GINI values and employing logistic regression for saliva and support vector machine models for stool, we successfully developed a microbiota test with heightened sensitivity and specificity for early CRC detection.

Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

Chronic activation of tubulin tyrosination in HCM mice and human iPSC-engineered heart tissues improves heart function.

Rationale: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that -α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Acute reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment. Objective: In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model ( Mybpc3 -knock-in; KI), in human HCM cardiomyocytes and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results: AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents and 3-week-old KI mice and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show that i) TTL for 6 weeks dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL for 12 weeks improved diastolic filling, cardiac output and stroke volume and reduced stiffness in KI mice; iii) TTL for 10 days normalized cell hypertrophy in HCM hiPSC-cardiomyocytes; iv) TTL induced a marked transcription and translation of several tubulins and modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-deficient EHTs exhibited reduced dTyr-tub levels, higher force and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion: This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

Application of sample displacement batch chromatography for fractionation of proteoforms.

Fractionation of proteoforms is currently the most challenging topic in the field of proteoform analysis. The need for considering the existence of proteoforms in experimental approaches is not only important in Life Science research in general but especially in the manufacturing of therapeutic proteins (TPs) like recombinant therapeutic antibodies (mAbs). Some of the proteoforms of TPs have significantly decreased actions or even cause side effects. The identification and removal of proteoforms differing from the main species, having the desired action, is challenging because the difference in the composition of atoms is often very small and their concentration in comparison to the main proteoform can be low. In this study, we demonstrate that sample displacement batch chromatography (SDBC) is an easy-to-handle, economical, and efficient method for fractionating proteoforms. As a model sample a commercial ovalbumin fraction was used, containing many ovalbumin proteoforms. The most promising parameters for the SDBC were determined by a screening approach and applied for a 10-segment fractionation of ovalbumin with cation exchange chromatography resins. Mass spectrometry of intact proteoforms was used for characterizing the SDBC fractionation process. By SDBC, a significant separation of different proteoforms was obtained.

Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex.

Here, we present a protocol for differential multi-omic analyses of distinct cell types in the developing mouse cerebral cortex. We describe steps for in utero electroporation, subsequent flow-cytometry-based isolation of developing mouse cortical cells, bulk RNA sequencing or quantitative liquid chromatography-tandem mass spectrometry, and bioinformatic analyses. This protocol can be applied to compare the proteomes and transcriptomes of developing mouse cortical cell populations after various manipulations (e.g., epigenetic). For complete details on the use and execution of this protocol, please refer to Meka et al. (2022).1.

Integrated proteomics spotlight the proteasome as a therapeutic vulnerability in embryonal tumors with multilayered rosettes.

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are rare malignant embryonal brain tumors. The prognosis of ETMR is poor and novel therapeutic approaches are desperately needed. Comprehension of ETMR tumor biology is currently based on only few previous molecular studies, which mainly focused on the analyses of nucleic acids. In this study, we explored integrated ETMR proteomics. METHODS: Using mass spectrometry, proteome data were acquired from 16 ETMR and the ETMR cell line BT183. Proteome data were integrated with case-matched global DNA methylation data, publicly available transcriptome data, and proteome data of further embryonal and pediatric brain tumors. RESULTS: Proteome-based cluster analyses grouped ETMR samples according to histomorphology, separating neuropil-rich tumors with neuronal signatures from primitive tumors with signatures relating to stemness and chromosome organization. Integrated proteomics showcased that ETMR and BT183 cells harbor proteasome regulatory proteins in abundance, implicating their strong dependency on the proteasome machinery to safeguard proteostasis. Indeed, in vitro assays using BT183 highlighted that ETMR tumor cells are highly vulnerable toward treatment with the CNS penetrant proteasome inhibitor Marizomib. CONCLUSIONS: In summary, histomorphology stipulates the proteome signatures of ETMR, and proteasome regulatory proteins are pervasively abundant in these tumors. As validated in vitro, proteasome inhibition poses a promising therapeutic option in ETMR.

Direct 3D Sampling of the Embryonic Mouse Head: Layer-wise Nanosecond Infrared Laser (NIRL) Ablation from Scalp to Cortex for Spatially Resolved Proteomics.

Common workflows in bottom-up proteomics require homogenization of tissue samples to gain access to the biomolecules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition, and rare cell types are not sufficiently represented. To overcome this problem, small-volume sampling and spatial resolution are needed to maintain a better representation of the sample composition and their proteome signatures. Using nanosecond infrared laser ablation, the region of interest can be targeted in a three-dimensional (3D) fashion, whereby the spatial information is maintained during the simultaneous process of sampling and homogenization. In this study, we ablated 40 µm thick consecutive layers directly from the scalp through the cortex of embryonic mouse heads and analyzed them by subsequent bottom-up proteomics. Extra- and intracranial ablated layers showed distinct proteome profiles comprising expected cell-specific proteins. Additionally, known cortex markers like SOX2, KI67, NESTIN, and MAP2 showed a layer-specific spatial protein abundance distribution. We propose potential new marker proteins for cortex layers, such as MTA1 and NMRAL1. The obtained data confirm that the new 3D tissue sampling and homogenization method is well suited for investigating the spatial proteome signature of tissue samples in a layerwise manner. Characterization of the proteome composition of embryonic skin and bone structures, meninges, and cortex lamination in situ enables a better understanding of molecular mechanisms of development during embryogenesis and disease pathogenesis.

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.

Proteome Analysis of Thyroid Hormone Transporter Mct8/Oatp1c1-Deficient Mice Reveals Novel Dysregulated Target Molecules Involved in Locomotor Function.

Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Expression Profiling along the Murine Intestine: Different Mucosal Protection Systems and Alterations in Tff1-Deficient Animals.

Tff1 is a typical gastric peptide secreted together with the mucin, Muc5ac. Tff1-deficient (Tff1KO) mice are well known for their prominent gastric phenotype and represent a recognized model for antral tumorigenesis. Notably, intestinal abnormalities have also been reported in the past in these animals. Here, we have compared the expression of selected genes in Tff1KO mice and their corresponding wild-type littermates (RT-PCR analyses), focusing on different mucosal protection systems along the murine intestine. As hallmarks, genes were identified with maximum expression in the proximal colon and/or the duodenum: Agr2, Muc6/A4gnt/Tff2, Tff1, Fut2, Gkn2, Gkn3, Duox2/Lpo, Nox1. This is indicative of different protection systems such as Tff2/Muc6, Tff1-Fcgbp, gastrokines, fucosylation, and reactive oxygen species (ROS) in the proximal colon and/or duodenum. Few significant transcriptional changes were observed in the intestine of Tff1KO mice when compared with wild-type littermates, Clca1 (Gob5), Gkn1, Gkn2, Nox1, Tff2. We also analyzed the expression of Tff1, Tff2, and Tff3 in the pancreas, liver, and lung of Tff1KO and wild-type animals, indicating a cross-regulation of Tff gene expression. Furthermore, on the protein level, heteromeric Tff1-Fcgbp and various monomeric Tff1 forms were identified in the duodenum and a high-molecular-mass Tff2/Muc6 complex was identified in the proximal colon (FPLC, proteomics).