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BioRxiv : the Preprint Server For... Jun 2024The retromer complex mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a multisubunit protease that...
The retromer complex mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a multisubunit protease that cleaves the transmembrane domain of its target proteins. Mutations in genes encoding subunits of retromer or γ-secretase can cause familial Alzheimer disease (AD) and other degenerative neurological diseases. It has been reported that retromer interacts with γ-secretase, but the consequences of this interaction are not known. Here, we report that retromer-mediated retrograde protein trafficking in cultured human epithelial cells is impaired by inhibition of γ-secretase activity or by genetic elimination of γ-secretase. γ-secretase inhibitor XXI and knockout of PS1, the catalytic subunit of γ-secretase, inhibit endosome to TGN trafficking of retromer-dependent retrograde cargos, divalent metal transporter 1 isoform II (DMT1-II), cation-independent mannose-6-phosphate receptor (CIMPR), and shiga toxin. Trafficking of retromer-independent cargos, such as cholera toxin and a CIMPR mutant that does not bind to retromer was not affected by γ-secretase inhibition. XXI treatment and PS1 KO inhibit interaction of γ-secretase with retromer but do not inhibit the association of cargo with retromer or with γ-secretase in intact cells. Similarly, these treatments do not affect the level of Rab7-GTP, which regulates retromer-cargo interaction. These results suggest that the γ-secretase-retromer interaction facilitates retromer-mediated retrograde trafficking.
PubMed: 38895404
DOI: 10.1101/2024.06.07.597932 -
BioRxiv : the Preprint Server For... Jun 2024Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In...
Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multi-subunit chromatin remodeling complex, ACF, where we also show protein transposition can occur within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.
PubMed: 38895383
DOI: 10.1101/2024.06.03.597171 -
BioRxiv : the Preprint Server For... Jun 2024RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small...
RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.
PubMed: 38895345
DOI: 10.1101/2024.06.08.598009 -
BioRxiv : the Preprint Server For... Jun 2024Adaptor protein complex 3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 is in a...
Adaptor protein complex 3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 is in a constitutively open, active conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted the core of human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy (cryo-EM). Similar to yeast AP-3, human AP-3 is in a constitutively open conformation, with the cargo-binding domain of the μ3 subunit conformationally free. To reconstitute AP-3 activation by the small GTPase Arf1, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures that show the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane-recruitment is driven by one of two predicted Arf1 binding sites on AP-3. In this conformation, AP-3 is flexibly tethered to the membrane and its cargo binding domain remains conformationally dynamic. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3 mediated vesicle coat assembly.
PubMed: 38895279
DOI: 10.1101/2024.06.05.597630 -
BioRxiv : the Preprint Server For... Jun 2024Mutations in gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX...
Mutations in gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2α phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2α phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation.
PubMed: 38895245
DOI: 10.1101/2024.06.04.597421 -
International Journal of Nanomedicine 2024Atypical teratoid rhabdoid tumor (ATRT) is a deadly, fast-growing form of pediatric brain cancer with poor prognosis. Most ATRTs are associated with inactivation of...
PURPOSE
Atypical teratoid rhabdoid tumor (ATRT) is a deadly, fast-growing form of pediatric brain cancer with poor prognosis. Most ATRTs are associated with inactivation of SMARCB1, a subunit of the chromatin remodeling complex, which is involved in developmental processes. The recent identification of SMARCB1 as a tumor suppressor gene suggests that restoration of SMARCB1 could be an effective therapeutic approach.
METHODS
We tested SMARCB1 gene therapy in SMARCB1-deficient rhabdoid tumor cells using a novel tumor-targeted nanomedicine (termed scL-SMARCB1) to deliver wild-type SMARCB1. Our nanomedicine is a systemically administered immuno-lipid nanoparticle that can actively cross the blood-brain barrier via transferrin receptor-mediated transcytosis and selectively target tumor cells via transferrin receptor-mediated endocytosis. We studied the antitumor activity of the scL-SMARCB1 nanocomplex either as a single agent or in combination with traditional treatment modalities in preclinical models of SMARCB1-deficient ATRT.
RESULTS
Restoration of SMARCB1 expression by the scL-SMARCB1 nanocomplex blocked proliferation, and induced senescence and apoptosis in ATRT cells. Systemic administration of the scL-SMARCB1 nanocomplex demonstrated antitumor efficacy as monotherapy in mice bearing ATRT xenografts, where the expression of exogenous SMARCB1 modulates MYC-target genes. scL-SMARCB1 demonstrated even greater antitumor efficacy when combined with either cisplatin-based chemotherapy or radiation therapy, resulting in significantly improved survival of ATRT-bearing mice.
CONCLUSION
Collectively, our data suggest that restoring SMARCB1 function via the scL-SMARCB1 nanocomplex may lead to therapeutic benefits in ATRT patients when combined with traditional chemoradiation therapies.
Topics: Animals; SMARCB1 Protein; Rhabdoid Tumor; Genetic Therapy; Mice; Cell Line, Tumor; Nanoparticles; Nanomedicine; Humans; Brain Neoplasms; Disease Models, Animal; Teratoma; Cell Proliferation; Antineoplastic Agents; Liposomes
PubMed: 38895149
DOI: 10.2147/IJN.S458323 -
PNAS Nexus Jun 2024Placental System L amino acid transporter activity is decreased in pregnancies complicated by intrauterine growth restriction (IUGR) and increased in fetal overgrowth....
Placental System L amino acid transporter activity is decreased in pregnancies complicated by intrauterine growth restriction (IUGR) and increased in fetal overgrowth. However, it is unknown if changes in the expression/activity of placental Large Neutral Amino Acid Transporter Small Subunit 1 (Slc7a5/LAT1) are mechanistically linked to placental function and fetal growth. We hypothesized that trophoblast-specific overexpression increases placental transport of essential amino acids, activates the placental mechanistic target of rapamycin (mTOR) signaling, and promotes fetal growth in mice. Using lentiviral transduction of blastocysts with a transgene, we achieved trophoblast-specific overexpression of OX) with increased fetal (+27%) and placental weights (+10%). Trophoblast-specific overexpression increased trophoblast plasma membrane (TPM) LAT1 protein abundance and TPM System L transporter (+53%) and System A transporter activity (+ 21%). overexpression also increased transplacental transport of leucine (+ 85%) but not of the System A tracer, 14C-methylamino isobutyric acid, in vivo. Trophoblast-specific overexpression of activated placental mTORC1, as assessed by increased (+44%) phosphorylation of S6 ribosomal protein (Ser 235/236), and mTORC2 as indicated by phosphorylation of PKCα-Tyr-657 (+47%) and Akt-Ser 473 (+96%). This is the first demonstration that placental transport of essential amino acids is mechanistically linked to fetal growth. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter in some cases of fetal overgrowth may directly contribute to the development of these pregnancy complications.
PubMed: 38894879
DOI: 10.1093/pnasnexus/pgae207 -
Molecules (Basel, Switzerland) Jun 2024Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold...
Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.
Topics: TRPM Cation Channels; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Signal Transduction; Phosphotransferases (Alcohol Group Acceptor); Transcription Factor AP-1; HEK293 Cells; Proto-Oncogene Proteins c-jun; Animals
PubMed: 38893478
DOI: 10.3390/molecules29112602 -
Cancers May 2024AT-rich interaction domain 1 () is a pivotal gene with a significant role in gastrointestinal tumors which encodes a protein referred to as BAF250a or SMARCF1, an... (Review)
Review
AT-rich interaction domain 1 () is a pivotal gene with a significant role in gastrointestinal tumors which encodes a protein referred to as BAF250a or SMARCF1, an integral component of the SWI/SNF (SWItch/sucrose non-fermentable) chromatin remodeling complex. This complex is instrumental in regulating gene expression by modifying the structure of chromatin to affect the accessibility of DNA. Mutations in have been identified in various gastrointestinal cancers, including colorectal, gastric, and pancreatic cancers. These mutations have the potential to disrupt normal SWI/SNF complex function, resulting in aberrant gene expression and potentially contributing to the initiation and progression of these malignancies. mutations are relatively common in gastric cancer, particularly in specific adenocarcinoma subtypes. Moreover, such mutations are more frequently observed in specific molecular subtypes, such as microsatellite stable (MSS) cancers and those with a diffuse histological subtype. Understanding the presence and implications of mutations in GC is of paramount importance for tailoring personalized treatment strategies and assessing prognosis, particularly given their potential in predicting patient response to novel treatment strategies including immunotherapy, poly(ADP) ribose polymerase (PARP) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitors.
PubMed: 38893181
DOI: 10.3390/cancers16112062 -
International Journal of Molecular... Jun 2024Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases....
Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.
Topics: Diabetic Retinopathy; Animals; Mice; Acetylglucosamine; N-Acetylglucosaminyltransferases; Diabetes Mellitus, Experimental; Humans; AMP-Activated Protein Kinases; Male; Apoptosis; Metformin; beta-N-Acetylhexosaminidases; Retina; Mice, Inbred C57BL; Cell Line
PubMed: 38892474
DOI: 10.3390/ijms25116286