International Journal of Molecular Sciences

Assessing autophagy promises to provide valuable information regarding the pathogenesis of chronic obstructive pulmonary disease (COPD). However, measuring the dynamic aspect of autophagy is challenging, and sample manipulation can cause signal fluctuations that deviate from the situation in vivo. Our aim was to assess an organotypic method to quantify autophagy in the context of COPD, where autophagy has demonstrated disease-related modulation. Blood from control and COPD participants was treated with/out chloroquine. Protein from peripheral blood mononuclear cells (PBMC) was then isolated and compared for LC3B-II abundance. Our observations show that while basal level LC3B-II abundance was similar between each group (P = 0.60), autophagic flux was significantly lower in the COPD cohort, suggesting disruption in autophagy (P = 0.004). We aim to extend this inquiry and compare pulmonary vs blood samples, to identify the utility of measuring autophagy in blood as a diagnostic outcome predictive of early COPD.

Pre-eclampsia (PE) is a hypertensive pregnancy complication. Oxidative stress is hypothesized to contribute to the pathophysiology of PE. Both the hydrogen sulfide (H2S) and oxytocin (OT) systems might play a role in the pathophysiology of PE, liked to their anti-oxidant and hypotensive effects. Thus, the role of the interaction of the OT and H2S systems in the context of PE was further elucidated in the present clinical case control study "NU-HOPE" (Nurnberg-Ulm: The role of H2S and Oxytocin Receptor in Pre-Eclampsia; ethical approval by the Landesarztekammer Bayern, file number 19033, 29th August 2019), comparing uncomplicated pregnancies, early-onset PE (ePE, onset <34 weeks gestational age) and late-onset PE (lPE, onset >34 weeks gestational age). Routine clinical data, serum H2S and homocysteine levels, tissue protein expression as well as nitrotyrosine formation were determined. The main findings were (i) unchanged plasma sulfide levels (ii) significantly elevated homocysteine levels in ePE, but not lPE, (iii) significantly elevated expression of H2S enzymes and OT receptor in the placenta in lPE, (iv) significantly elevated nitrotyrosine formation in the lPE myometrium. Taken together, these findings suggest a role for the interaction of the endogenous H2S- and OT/OTR systems in the pathophysiology of pre-eclampsia, possibly linked to impaired antioxidant protection.

DNA repair pathways are critical processes that need both spatial and temporal fine regulation. Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions, while excluding non-interacting components. Proteins disordered domains, as well as RNA, favor condensation to modulate this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses. In this context, it is emerging that RNA-processing pathways and PARylation events, through the addition of an ADP-ribose moiety to both proteins and DNA, participate in different aspects of the DNA Damage Response (DDR). Remarkably, defects in these regulatory connections are associated with genomic instability and human pathologies. In addition, it has been recently noticed that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway of non-distorting DNA lesions, bearing additional non-canonical DNA-repair functions associated with processes coping with RNA metabolism. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in several tumorigenic and amyloidogenic events. This work represents a paradigmatical pipeline for evaluating the relevance of LLPS in DDR. Statement of significanceIn this work, we aimed to test the hypothesis of an involvement of phase-separation in regulating the molecular mechanisms of the multifunctional enzyme APE1 starting from the analysis of its recently-characterized protein-protein interactome (PPI). We compared APE1-PPI to phase-separation databases and we performed functional enrichment analysis, uncovering links between APE1 and already known demixing factors, establishing an association with liquidliquid phase separation. This analysis could represent a starting point for implementing downstream experimental validations, using in vitro and in vivo approaches, to assess actual demixing.

Macrophages are required for our bodys development and tissue repair and protect against microbial attacks. We previously reported a crucial role for regulation of mRNA 3-end cleavage and polyadenylation (C/P) in monocyte to macrophage differentiation. The CFIm25 subunit of the C/P complex showed a striking increase upon differentiation of monocytes with Phorbol Myristate Acetate, suggesting that it promotes this process. To test this hypothesis, CFIm25 was overexpressed in two different monocytic cell lines, followed by differentiation. Both cell lines showed a significant increase in macrophage characteristics and an earlier slowing of the cell cycle. In contrast, depletion of CFIm25 hindered differentiation. Cell cycle slowing upon CFIm25 overexpression was consistent with a greater decrease in the proliferation markers PCNA and cyclin D1, coupled with increased 3UTR lengthening of cyclin D1 mRNA. Since choice of other poly(A) sites could be affected by manipulating CFIm25, we identified additional genes with altered use of poly(A) sites during differentiation and examined how this changed upon CFIm25 overexpression. The mRNAs of positive regulators of NF-{kappa}B signaling, TAB2 and TBL1XR1, and NFKB1, which encodes the NF-{kappa}B p50 precursor, underwent 3UTR shortening that was associated with increased protein expression compared to the control. Cells overexpressing CFIm25 also showed elevated levels of phosphorylated NF-{kappa}B-p65 and the NF-{kappa}B targets p21, Bcl-XL, ICAM1 and TNF- at an earlier time and greater resistance to NF-{kappa}B chemical inhibition. In conclusion, our study supports a model in which CFIm25 accelerates the monocyte to macrophage transition by promoting alternative polyadenylation events which lead to activation of the NF-{kappa}B pathway.

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

Induction of unfolded protein response (UPR) involves activation of transcription factor Hac1p that facilitates the transactivation of genes encoding ER-chaperones. Hac1p is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE) at its 3'-UTR. This precursor RNA undergoes a reversible and differential intra-nuclear mRNA decay by the nuclear exosome/CTEXT at various phases of UPR. In this investigation, using a combination of genetic, and biochemical approach, the Rab-GTPase Ypt1p is demonstrated to control UPR signaling dynamics. Regulation of UPR by Ypt1p relies on its characteristic nuclear localization in absence of ER-stress resulting in its strong association with pre-HAC1 mRNA at its 3'-UTR that promotes sequential recruitments of Nrd1-Nab3p-Sen1p (NNS) complex [-&gt;] CTEXT [-&gt;] the nuclear exosome onto the pre-HAC1 mRNA that is accompanied by its rapid and selective nuclear decay. This accelerated 3'[-&gt;]5' mRNA decay produces a pre-HAC1 mRNA pool lacking the functional BE thus causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers a rapid relocalization of Ypt1p to the cytoplasm with its consequent dissociation from pre-HAC1 mRNA thereby causing a decreased recruitment of NNS/exosome/CTEXT to precursor HAC1 RNA leading to its diminished 3'[-&gt;]5' degradation by the exosome. This diminished decay produces an increased abundance of pre-HAC1 mRNA population with intact functional BE leading to its enhanced recruitment to Ire1p foci that is followed by its increased splicing and translation. This enhanced translation produces a huge burst of Hac1p that rapidly transactivates the genes encoding ER-chaperones.

Tristetraprolin (TTP)/ ZFP36 is an RNA binding protein that is involved in the turnover regulation of target adenylate-uridylate-rich RNA element (ARE) containing mRNAs. AREs are present in the 3-untranslated region of transcripts expressed from many immediate-early genes, including cytokines and chemokines. It has been demonstrated that TTP-mediated post-transcriptional mRNA decay regulation is crucial for modulating physiological control, particularly in response to inflammatory stimulation. TTP is associated with the CCR4-NOT deadenylation complex through the TTP C-terminus to promote mRNA decay. However, it is not fully understood whether there are additional sites within TTP that contribute to its function through interacting with other factors. We analyzed the functionality of the unique tryptophan residues located in the TTP N-terminus using a cell-based assay system that consists of a tetracycline-responsive CMV promoter-driven, intron-inserted luciferase (LUC). This system enabled us to analyze TTP activity during both the early phase and the steady-state phase of gene expression, as well as in the post-transcriptional mRNA decay following the treatment of tetracycline analogs. Meanwhile, we identified putative TTP associates using a proximity labeling method. We found that tryptophan residues in the TTP N-terminus together with CNOT10, a component of the CCR4-NOT complex, were involved specifically in the reduction of the ARE-containing LUC mRNA level during the early phase of gene expression. However, they were not involved in the decay of LUC mRNA in the steady-state phase. We propose a novel post-transcriptional TTP functionality in the reduction of ARE-containing mRNA level, which differs from the well-characterized mRNA decay activity.

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Its own regulation is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the Ddx5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We postulate that its range of interactions with other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1. These individual interactions provide quantitative contributions that, in sum, establish high level activity of the KLF1 promoter and suggest they can be selectively manipulated for clinical benefit.

Overactive bladder (OAB) is a urological symptom complex defined by urinary urgency. It can have a devastating impact on an individuals quality of life and leads to significant financial cost. Insulin-like growth factor 1 (IGF-1) is a protein hormone involved in a broad range of processes including cell proliferation and differentiation. IGF-1 is also regulated through alternative splicing. While the primary IGF-1Ea transcript is highly expressed in liver, the alternative IGF-1Ec transcript encodes the proteolytically-derived MGF peptide and has been primarily studied in skeletal muscle. MGF has been shown to stimulate satellite cell proliferation following tissue mechanical stretch or injury, but the role of MGF in smooth muscle, such as the detrusor muscle of the bladder, has been little explored. The aim of this study was to explore the expression of MGF in bladder biopsies from patients with OAB and age-matched controls. We show using immunohistochemistry that MGF is widely expressed in bladder tissue. Quantification of MGF expression by western blot showed that average MGF expression is more than doubled in OAB biopsies compared to controls (mean MGF in OAB=0.51{+/-}0.1, n=23; mean MGF in controls=0.22{+/-}0.07, n=9; p=0.05). Furthermore, there is an inverse correlation between MGF protein levels and symptom severity, as determined by the urodynamic parameter maximum cystometric capacity (correlation=0.53, p=0.03 n=16). MGF expression was highest in OAB biopsies with strong expression of the muscle cell marker DES. Combined with our observation that MGF induces cell proliferation in primary bladder cultures, our data suggests that high MGF expression in OAB patients may represent an attempted protective response in the bladder.

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

CFTR misfolding due to cystic fibrosis causing mutations can be corrected with small molecules designated as correctors. VX-809, an investigational corrector compound, is believed to bind CFTR directly to either the first membrane-spanning domain (MSD1) and/or the first nucleotide-binding domain (NBD1). Blind docking onto the 3D structures of these domains, followed by molecular dynamics (MD) simulations, revealed the presence of two potential VX-809 binding sites which, when mutated, abrogated corrector rescue. Mutations altering protein maturation are also shown to be not equally sensitive to the occupancy of the two sites by VX-809, with the most frequent mutation F508del requiring integrity of both sites and allosteric coupling with the F508del region while L206W only requires the integrity of the MSD1 site. A network of charged amino acids in the lasso Lh2 helix and the intracellular loops ICL1 and ICL4 is involved in the allostery between MSD1 and NBD1. Corrector VX-445, which is used in combination in clinics with VX-661, a structurally close analog of VX-809, to fully correct F508del, is also shown to occupy two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. In conclusion, VX-809 and VX-445 appear to bind different CFTR domains to alleviate specific folding defects. These results provide new insights into therapeutics understanding and may help the development of efficient corrector combinations.

Osteoarthritis (OA) is the most prevalent disorder of articulating joints and a leading cause of disability in humans, affecting half of the worlds population aged 65 years or older. Articular cartilage and synovial tissue from OA patients show an overactivity of the membrane channel protein connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration. We have recently demonstrated the use of the Cx43 as an appropriate therapeutic target to halt OA progression by decreasing the accumulation of senescent cells and by triggering redifferentiation of osteoarthritic chondrocytes (OACs) into a more differentiated state, restoring the fully mature phenotype and cartilage regeneration. In this study we have found that small molecular polyphenols derived by olive extracts target Cx43 and senescence in OACs, synovial and bone cells from patients and in human mesenchymal stem cells (hMSCs). Our results indicate that these small molecules including oleuropein regulate the promoter activity of Cx43 gene. The downregulation of Cx43 expression by oleuropein reduce gap junction intercellular communication, cellular senescence in chondrocytes and enhance the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. In concordance with these results, these small molecules reduce Cx43 and decrease Twist-1 activity leading to redifferentiation of OACs, which restores the synthesis of cartilage ECM components (Col2A1 and proteoglycans) and reduces inflammatory and catabolic factors IL-1{beta}, IL-6, COX-2 and MMP-3 and cellular senescence orchestrated by p53/p21 together with the synthesis of SASP via NF-kB. Altogether, our results demonstrate the use of the olive-derived polyphenols such as oleuropein as potentially effective therapeutic agents to enhance the efficacy of hMSC therapy and to induce a pro-regenerative environment in OA patients by restoring cellular phenotype and clearing out senescent cells in joint tissues in order to stop or prevent the progression of the disease.

Treatment of endothelial cells with bacterial lipopolysaccharide (LPS) evokes a number of metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Here effects of LPS on human aortic endothelial cells (HAEC) have been investigated. Among the spectrum of biochemical changes substantially elevated N-nicotinamide methyltransferase (NNMT) protein level was particularly intriguing. This important enzyme may potentially affect cellular metabolism by two means: direct regulation of methylnicotinamide level and availability of nicotinamide, that at least potentially may influence NAD+ synthesis, and regulation of S-adenosylmethionine concentration and therefore controlling methylation of many proteins including chromatin. This may have epigenetic consequences. This paper is focused on NNMT, despite the fact that in the presence of LPS additional effects of this compound mask pure (canonical) consequences of the elevated NNMT protein which are an increased MNA synthesis or reduced NAD+ level. On the other hand, however, it has been shown that silencing of the NNMT-encoding gene prevents several changes which are observed in control HAECs treated with LPS. They include significantly increased calcium response to thapsigargin (store-operated calcium entry), altered energy metabolism which is switched to anaerobic glycolysis and rearrangement of the mitochondrial network. However, a biochemical mechanism behind the protective consequences of the NNMT deficiency in cells treated with LPS remains unexplained.

Every cell has a multifaceted phenotype. Transcriptional analysis of functionally defined groups of genes can provide insight into this phenotypic complexity. In the present study, the mRNA transcriptome of phorbol ester (TPA) differentiated HL-60/S4 macrophage cells was scrutinized using Gene Set Enrichment Analysis (GSEA), which evaluates the strengths of various cellular phenotypes by examining the enrichment of functionally different gene sets. Employing GSEA, we obtained supporting evidence that HL-60/S4 macrophages are senescent, probably a consequence of enriched TGF{beta} and NOTCH signaling transcripts. There appears to be a reduction of transcripts for heterochromatin, nucleosome formation, and chromatin remodeling phenotypes. In addition, despite upregulated oxidative stress gene transcription, we observed a reduction of DNA damage and repair transcripts. GSEA indicated that transcripts for autophagy, extracellular matrix, and inflammation/inflammasomes are enriched. We also observed that the HL-60/S4 macrophage is enriched for apoptosis gene transcripts, which may promote necrotic death by pyroptosis. The long-term goal of this research direction is to see whether this complex multifaceted phenotypic pattern is shared with other types of macrophages and to determine what mechanisms might exist to coordinate these phenotypic facets within a single cell.

The YODA kinase (YDA) pathway is intimately associated with the control of Arabidopsis thaliana embryo development but little is known regarding its regulators. Using genetic analysis, HEAT SHOCK PROTEINS 90 (HSP90s) emerge as potent regulators of YDA in the process of embryo development and patterning. This study is focused on the characterization and quantification of early embryonal traits of single and double hsp90 and yda mutants. The mutant analysis was supported by expression analyses of cell-specific WUSCHEL-RELATED HOMEOBOX 2 (WOX2) and WOX8 genes during early embryonic development. Chromatin immunoprecipitation assays corroborated the involvement of YDA and HSP90s in the epigenetic control of chromatin remodeling during early embryogenesis. Genetic interactions among HSP90s and members of the YDA signaling pathway affected the development of both embryo proper and suspensor. Impaired function of HSP90s or YDA had an impact on the spatiotemporal expression of WOX8 and WOX2 suggesting their essential role in cell fate determination and interference with auxin distribution. Hence, the interplay between HSP90s and YDA signaling cascade mediates the epigenetic control regulating the transcriptional networks shaping early embryo development.

The adenosine 2a receptor (A2aR) is a G-protein coupled receptor that has important anti-inflammatory effects in response to some agonists and consequently is considered a therapeutic target. Its activity is affected by local membrane lipid environment and presence of certain phospholipid classes, so studies should be conducted using extraction methods such as styrene maleic acid co-polymers (SMA) that retain the local lipids. Currently, little is known about the effect of oxidative stress, which may arise from inflammation, on the A2aR. Therefore it was over-expressed in Pichia pastoris, SMA was used to extract the A2aR from cell membranes and its response to ligands was tested in the presence or absence of the radical initiator AAPH or reactive aldehyde acrolein. SMA-extracted A2aR was able to undergo conformational changes, measured by tryptophan fluorescence, in response to its ligands but oxidative treatments had no effect on the structural changes. Similarly, the treatments did not affect temperature-dependent protein unfolding. In contrast, in HEK293 cells expressing the A2aR, oxidative treatments increased cAMP levels in response to the agonist NECA, independently of adenylate cyclase activity. Thus, oxidative stress may be a homeostatic mechanism that abrogates inflammation via the A2aR signalling pathway. (max. 200 words - 194)

The prefoldin complex (PFDc) participates in cellular proteostasis in eukaryotes by acting as cochaperone of the chaperonin CTT. This role is mainly exerted in the cytoplasm where it contributes to the correct folding of client proteins, thus preventing them to form aggregations and cellular damage. Several reports indicate, however, that they also play a role in transcriptional regulation in the nucleus in several model species. In this work, we have investigated how extended is the role of PFDs in nuclear processes by inspecting their interactome and their coexpression networks in yeast, fly, and humans. The analysis indicates that they may perform extensive, conserved functions in nuclear processes. The construction of the predicted interactome for Arabidopsis PFDs, based on the ortholog interactions, has allowed us to identify many putative PFD interactors linking them to unanticipated processes, such as chromatin remodeling. Based on this analysis, we have investigated the role of PFDs in H2A.Z deposition through their interaction with the chromatin remodeling complex SWR1c. Our results show that PFDs have a positive effect on SWR1c, which is reflected in defects in H2A.Z deposition in hundreds of genes in seedlings defective in PFD3 and PFD5 activities.

The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with HSP70 and HSPB8. Under aging and cellular stress conditions in particular, together with molecular chaperones, BAG3 ensures the sequestration of aggregated or aggregation prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. There are emerging indications that BAG3-mediated selective macroautophagy also copes with non-ubiquitinated cargo. Phylogenetically, BAG3 comprises several highly conserved predicted LIRs, LC3-interacting regions, which might directly target BAG3 including its cargo to ATG8 proteins and directly drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with ATG8 proteins. In addition, we could demonstrate that mutations within the LIRs impair co-localization with ATG8 proteins in immunofluorescence. A BAG3 variant mutated in all LIRs results in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared to wild-type BAG3. These results strongly suggest LC3-mediated sequestration of BAG3. Therefore, we conclude that in addition of being a key co-chaperone to HSP70, BAG3 may also act as cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control. SynopsisBAG3 ensures sequestration of aggregated ubiquitinated proteins to the autophagic-lysosomal degradation. Based on emerging indications this BAG3-mediated macroautophagy may also cope with non-ubiquitinated clients and comprises conserved predicted LC3 interacting regions, we analyzed the interaction with LC3 proteins. We evidenced an interaction of BAG3 with LC3 proteins by various measures including pull-down experiments, peptide arrays, proximity ligation assays, co-localization and native autophagic vesicles analysis. These results suggest BAG3 may additionally act as cargo receptor for client proteins. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/526551v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@ac40d2org.highwire.dtl.DTLVardef@d3bf3dorg.highwire.dtl.DTLVardef@1b06a53org.highwire.dtl.DTLVardef@114a453_HPS_FORMAT_FIGEXP M_FIG C_FIG

Endometriosis is a gynecological disease characterized by the presence of uterine (eutopic) endometrial glands and tissues outside the intra-uterine locations, in ectopic regions such as the pelvic peritoneum, fallopian tubes, or ovaries. Approximately 5-10% of reproductive and 20-50% of infertile women are affected by endometriosis. The pathogenesis of endometriosis involves various factors, including hormonal, environmental, genetic, and immune system components, directly or indirectly altering estrogen levels and impacting womens reproductive health.This study aimed to identify novel and potential biomarkers for endometriosis using mRNA seq analysis. Differentially expressed genes (DEGs) were identified from raw gene expression profiles, and their functional analysis was subsequently conducted. A total of 552 DEGs (312 upregulated and 240 downregulated) were identified in samples from women with endometriosis compared to control subjects.Major DEGs, such as C3, PSAP, APP, GNG12, were identified as hub nodes and found to be involved in various functions, including epithelial cell differentiation and development, proteolysis, gland development, muscle fiber development, and response to hormone stimulus. These DEGs may play a direct or indirect role in the pathogenesis of endometriosis, serving as potential biomarkers for ectopic endometrium. While this study provides a preliminary insight into the mechanism of endometriosis, further detailed studies are necessary to fully understand its path of action.

The link between cholesterol homeostasis and the cleavage of the amyloid precursor protein (APP), and their relationship to the pathogenesis of Alzheimers disease (AD) is still unknown. Cellular cholesterol levels are regulated by a crosstalk between the plasma membrane (PM), where most of the cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol resides. This crosstalk between PM and ER is believed to be regulated by lipid-sensing peptide(s) that can modulate the internalization of extracellular cholesterol and/or its de novo synthesis in the ER. Our data here indicates that the 99-aa C-terminal fragment of APP (C99), a cholesterol-binding peptide, regulates cholesterol trafficking between the PM and the ER. In AD models, increases in C99 provoke the upregulation of cholesterol internalization and its delivery to the ER, which in turn result into the loss of lipid homeostasis and the appearance of AD signatures, such as higher production of longer forms of amyloid {beta}. Our data suggest a novel role of C99 as mediator of cholesterol disturbances in AD, and as a potential early hallmark of the disease.