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The COVID-19 pandemic has significantly affected both the global economy and human health. COVID-19 is a single-stranded RNA virus that spreads through the inhalation of viral droplets. Its genome encodes four structural proteins: the envelop protein, spike protein, membrane protein, and nucleocapsid protein. The virus's capsid is a protein shell that contains a positive RNA strand that allows it to take control of human cell machinery. Currently, the only treatments for COVID-19 are preventative measures and immunization. Some of the anti-COVID and anti-cancer medications used to combat this virus include Camptothecin Polymer, Hyaluronic Acid-curcumin, and Hydroxychloroquine. Graphs are used extensively in chemical investigation to provide a mathematical model of molecules. The selection of graph invariants, formerly known as molecular structure descriptor's, is the mathematical modelling of chemical compounds' physio-chemical, pharmacologic, and toxicological aspects. This paper presents a new technique to compute topological indices for three types of chemical networks for a few anti-COVID and anti-cancer medications. Our findings suggest that these indices show a strong association with the acentric factor and entropy, which make them effective in QSPR and QSAR analysis with high accuracy. [ABSTRACT FROM AUTHOR]

Cell-free synthesis systems can complete the transcription and translation process in vitro to produce complex proteins that are difficult to be expressed in traditional cell-based systems. Such systems also can be used for the assembly of efficient localized multienzyme cascades to synthesize products that are toxic to cells. Cell-free synthesis systems provide a simpler and faster engineering solution than living cells, allowing unprecedented design freedom. This paper reviews the latest progress on the application of cell-free synthesis systems in the field of enzymatic catalysis, including cell-free protein synthesis and cell-free metabolic engineering. In cell-free protein synthesis: complex proteins, toxic proteins, membrane proteins, and artificial proteins containing non-natural amino acids can be easily synthesized by directly controlling the reaction conditions in the cell-free system. In cell-free metabolic engineering, the synthesis of desired products can be made more specific and efficient by designing metabolic pathways and screening biocatalysts based on purified enzymes or crude extracts. Through the combination of cell-free synthesis systems and emerging technologies, such as: synthetic biology, microfluidic control, cofactor regeneration, and artificial scaffolds, we will be able to build increasingly complex biomolecule systems. In the next few years, these technologies are expected to mature and reach industrialization, providing innovative platforms for a wide range of biotechnological applications. [ABSTRACT FROM AUTHOR]

FACT is a developed technique for clearing tissues that does not use acrylamide. Since the removal of lipids is crucial for transparency and efficient antibody staining throughout the tissue, especially for microscopy and imaging, it is a harmful process that can cause the loss of important biological molecules such as proteins. The FACT technique overcomes this by chemically bonding the membrane and intracellular proteins with the extracellular matrix, creating a massive 3D hydrogel matrix and providing structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, the FACT technique requires less labor and harmful chemicals and is therefore considered a more suitable option. In this study, we describe the complete FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving structure and improving image quality. The protocol includes tissue perfusion, fixation, clearing, antibody staining, refractive index matching (RIM) (), microscopy, and imaging. The timing for each step varies depending on the size, weight, and type of tissue, as well as the type of immunostaining. We provide an example of the FACT protocol using mouse brain tissue, which demonstrates its suitability for molecular interrogation analysis of large tissues. The FACT technique has been successfully performed on different types of tissues, making it a favorable choice for a variety of applications. [ABSTRACT FROM AUTHOR]

In our recent paper, we uncovered that ATG3 exhibits a large degree of structural dynamics on autophagic membranes to efficiently carry out LC3 lipidation. ATG3 proteins possess an amphipathic α-helix (AH) identified by a small number of bulky and hydrophobic residues. This biophysical fingerprint allows for transient membrane association of ATG3 and facilitates its enzymatic reaction. This study will pave the way for a structural and mechanistic understanding of how membrane association of ATG proteins is orchestrated during autophagosome formation. [ABSTRACT FROM AUTHOR]

Platelet secretion requires Soluble N-ethylmaleimide Sensitive Attachment Protein Receptors (SNAREs). Vesicle SNAREs/Vesicle-Associated Membrane Proteins (v-SNAREs/VAMPs) on granules and t-SNAREs in plasma membranes mediate granule release. Platelet VAMP heterogeneity has complicated the assessment of how/if each is used and affects hemostasis. To address the importance of VAMP-7 (V7), we analyzed mice with global deletions of V3 and V7 together or platelet-specific deletions of V2, V3, and global deletion of V7. We measured the kinetics of cargo release, and its effects on three injury models to define the context-specific roles of these VAMPs. Loss of V7 minimally affected dense and a granule release but did affect lysosomal release. V3-/-7-/- and V2Δ3Δ7-/- platelets showed partial defects in a and lysosomal release; dense granule secretion was unaffected. In vivo assays showed that loss of V2, V3, and V7 caused no bleeding or occlusive thrombosis. These data indicate a role for V7 in lysosome release that is partially compensated by V3. V7 and V3, together, contribute to a granule release, however none of these deletions affected hemostasis/thrombosis. Our results confirm the dominance of V8. When it is present, deletion of V2, V3, or V7 alone or in combination minimally affects platelet secretion and hemostasis. [ABSTRACT FROM AUTHOR]

F11 receptor (F11R)/Junctional Adhesion Molecule -A (JAM-A) is a transmembrane protein which belongs to the immunoglobulin superfamily of cell adhesion molecules. F11R/JAM-A is present in epithelial cells, endothelial cells, leukocytes, and blood platelets. In epithelial and endothelial cells, it takes part in the formation of tight junctions. In these structures, molecules of F11R/JAM-A located on adjacent cells form homodimers and thus take part in stabilization of cellular layer integrity. In leukocytes, F11R/JAM-A was shown to play role in their transmigration through the vascular wall. Paradoxically, the function of F11R/JAM-A in blood platelets, where it was primarily discovered, is much less understood. It has been proven to regulate downstream signaling of aIIbß3 integrin and to mediate platelet adhesion under static conditions. It was also shown to contribute to transient interactions of platelets with inflamed vascular wall. The review is aimed at summarizing the current state of knowledge of the platelet pool of F11R/JAM-A. The article also presents perspectives of the future research to better understand the role of this protein in hemostasis, thrombosis, and other processes where blood platelets are involved. [ABSTRACT FROM AUTHOR]

The structural proteins located on the SARS-CoV-2 envelope, namely the spike, membrane and envelope protein, play important roles during the entire viral infection cycle. For example, the interaction between the membrane protein and the other structural proteins results in the formation of new virions, while the envelope protein then mediates their secretion. The membrane protein and envelope protein are considered potential drug targets, driving future exploration of their structures and interactions. However, research efforts are complicated as both proteins are membrane-bound and have several functions depending on their tertiary and quaternary structure. Here we review data originating from different sources and explain what is known about the structure and function of these proteins to date. [ABSTRACT FROM AUTHOR]

The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, has a major role in different types of pain. TRPA1 is primarily localized to a subpopulation of primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia. This subset of nociceptors produces and releases the neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP), which mediate neurogenic inflammation. TRPA1 is characterized by unique sensitivity for an unprecedented number of reactive byproducts of oxidative, nitrative, and carbonylic stress and to be activated by several chemically heterogenous, exogenous, and endogenous compounds. Recent preclinical evidence has revealed that expression of TRPA1 is not limited to neurons, but its functional role has been reported in central and peripheral glial cells. In particular, Schwann cell TRPA1 was recently implicated in sustaining mechanical and thermal (cold) hypersensitivity in mouse models of macrophage-dependent and macrophage-independent inflammatory, neuropathic, cancer, and migraine pain. Some analgesics and herbal medicines/natural products widely used for the acute treatment of pain and headache have shown some inhibitory action at TRPA1. A series of high affinity and selective TRPA1 antagonists have been developed and are currently being tested in phase I and phase II clinical trials for different diseases with a prominent pain component. Abbreviations: 4-HNE, 4-hydroxynonenal; ADH-2, alcohol dehydrogenase-2; AITC, allyl isothiocyanate; ANKTD, ankyrin-like protein with transmembrane domains protein 1; B2 receptor, bradykinin 2 receptor; CIPN, chemotherapeutic-induced peripheral neuropathy; CGRP, calcitonin gene related peptide; CRISPR, clustered regularly interspaced short palindromic repeats; CNS, central nervous system; COOH, carboxylic terminal; CpG, C-phosphate-G; DRG, dorsal root ganglia; EP, prostaglandins; GPCR, G-protein-coupled receptors; GTN, glyceryl trinitrate; MAPK, mitogen-activated protein kinase; M-CSF, macrophage-colony stimulating factor; NAPQI, N-Acetyl parabenzoquinone-imine; NGF, nerve growth factor; NH2, amino terminal; NKA, neurokinin A; NO, nitric oxide; NRS, numerical rating scale; PAR2, protease-activated receptor 2; PMA, periorbital mechanical allodynia; PLC, phospholipase C; PKC, protein kinase C; pSNL, partial sciatic nerve ligation; RCS, reactive carbonyl species; ROS, reactive oxygen species; RNS, nitrogen oxygen species; SP, substance P; TG, trigeminal ganglion; THC, Δ9-tetrahydrocannabinol; TrkA, neurotrophic receptor tyrosine kinase A; TRP, transient receptor potential; TRPC, TRP canonical; TRPM, TRP melastatin; TRPP, TRP polycystin; TRPM, TRP mucolipin; TRPA, TRP ankyrin; TRPV, TRP vanilloid; VG, vagal ganglion; [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs) are considered one of the most critical stromal cells that interact with pancreatic ductal adenocarcinoma (PDAC) and promote tumor growth, metastasis, and treatment resistance. Previous studies illustrated macroautophagy/autophagy contributes to CAF activation during tumor progression. Here in our study, we found that autophagy deficiency in CAFs impedes CAF activation by inhibiting proline biosynthesis and collagen production. Furthermore, we uncovered that autophagy promotes proline biosynthesis through mitophagy-mediated regulation of NADK2 (NAD kinase 2, mitochondrial), an enzyme responsible for production of mitochondrial NADP(H). Using an orthotopic mouse model of PDAC, we found that inhibiting mitophagy by targeting PRKN (parkin RBR E3 ubiquitin protein ligase) in the stroma reduced tumor weight. Thus, inhibition of CAFs mitophagy might be an attractive strategy for stroma-focused anti-cancer intervention. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; ALDH18A1/P5CS: aldehyde dehydrogenase 18 family, member A1; ATG3: autophagy related 3; ATG5: autophagy related 5; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CAFs:cancer-associated fibroblasts; COL1A1: collagen, type I, alpha 1; DES: desmin; ECM: extracellular matrix; FABP4: fatty acid binding protein 4, adipocyte; FAP/FAPα: fibroblast activation protein; IHC: immunohistochemical staining; LAMP1: lysosomal-associated membrane protein 1; NADK2: NAD kinase 2, mitochondrial; PC1: pro-collagen 1; PDAC: pancreatic ductal adenocarcinoma; PDGFR: platelet derived growth factor receptor; PDPN: podoplanin; PRKN: parkin RBR E3 ubiquitin protein ligase; PSCs: pancreatic stellate cells; VIM: vimentin; WT: wild-type [ABSTRACT FROM AUTHOR]

Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target. Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type. [ABSTRACT FROM AUTHOR]

Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose–response curve of the MOG transfected cell lysate coating plate can be 2–9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance. [ABSTRACT FROM AUTHOR]

Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AFSM: autofluorescent storage materials; ANCL: adult neuronal ceroid lipofuscinosis; Baf. A1: bafilomycin A1; CLN: ceroid lipofuscinosis neuronal; CLU: clusterin; CS: cysteine string domain of DNAJC5/CSPα; CUPS: compartment for unconventional protein secretion; DN: dominant negative; DNAJC5/CSPα: DnaJ heat shock protein family (Hsp40) member C5; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INCL: infant neuronal ceroid lipofuscinosis; JNCL: juvenile neuronal ceroid lipofuscinosis; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAPTM4B: lysosomal protein transmembrane 4 beta; LN: linker domain of DNAJC5/CSPα; MAPS: misfolding-associated protein secretion; mCh/Ch: mCherry; mCi/Ci: mCitrine; MTOR: mechanistic target of rapamycin kinase; NCL: neuronal ceroid lipofuscinosis; PPT1: palmitoyl-protein thioesterase 1; PQC: protein quality control; SBP: streptavidin binding protein; SGT: small glutamine-rich tetratricopeptide repeat; shRNA: short hairpin RNA; SLC3A2/CD98hc: solute carrier family 3 member 2; SNCA/α-synuclein: synuclein alpha; TMED10: transmembrane p24 trafficking protein 10; UV: ultraviolet; VPS4: vacuolar protein sorting 4 homolog; WT: wild type. [ABSTRACT FROM AUTHOR]

Selective macroautophagy/autophagy maintains cellular homeostasis through the lysosomal degradation of specific cellular proteins or organelles. The pro-survival effect of selective autophagy has been well-characterized, but the mechanism by which it drives cell death is still poorly understood. Here, we use a quantitative proteomic approach to identify HPCAL1 (hippocalcin like 1) as a novel autophagy receptor for the selective degradation of CDH2 (cadherin 2) during ferroptosis. HPCAL1-dependent CDH2 depletion increases susceptibility to ferroptotic death by reducing membrane tension and favoring lipid peroxidation. Site-directed mutagenesis aided by bioinformatic analyses revealed that the autophagic degradation of CDH2 requires PRKCQ (protein kinase C theta)-mediated HPCAL1 phosphorylation on Thr149, as well as a non-classical LC3-interacting region motif located between amino acids 46–51. An unbiased drug screening campaign involving 4208 small molecule compounds led to the identification of a ferroptosis inhibitor that suppressed HPCAL1 expression. The genetic or pharmacological inhibition of HPCAL1 prevented ferroptosis-induced tumor suppression and pancreatitis in suitable mouse models. These findings provide a framework for understanding how selective autophagy promotes ferroptotic cell death.Abbreviations: ANXA7: annexin A7; ARNTL: aryl hydrocarbon receptor nuclear translocator like; CCK8: cell counting kit-8; CDH2: cadherin 2; CETSAs: cellular thermal shift assays; CPT2: carnitine palmitoyltransferase 2; DAMP, danger/damage-associated molecular pattern; DPPH: 2,2-diphenyl-1-picrylhydrazyl; DFO: deferoxamine; EBNA1BP2: EBNA1 binding protein 2; EIF4G1: eukaryotic translation initiation factor 4 gamma 1; FBL: fibrillarin; FKBP1A: FKBP prolyl isomerase 1A; FTH1: ferritin heavy chain 1; GPX4: glutathione peroxidase 4; GSDMs: gasdermins; HBSS: Hanks' buffered salt solution; HMGB1: high mobility group box 1; HNRNPUL1: heterogeneous nuclear ribonucleoprotein U like 1; HPCAL1: hippocalcin like 1; H1-3/HIST1H1D: H1.3 linker histone, cluster member; IKE: imidazole ketone erastin; KD: knockdown; LDH: lactate dehydrogenase; LIR: LC3-interacting region; MAGOH: mago homolog, exon junction complex subunit; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehyde; MLKL: mixed lineage kinase domain like pseudokinase; MPO: myeloperoxidase; MTOR: mechanistic target of rapamycin kinase; OE: overexpressing; OSTM1: osteoclastogenesis associated transmembrane protein 1; PRKC/PKC: protein kinase C; PRKAR1A: protein kinase cAMP-dependent type I regulatory subunit alpha; PRDX3: peroxiredoxin 3; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; SLC40A1: solute carrier family 40 member 1; SPTAN1: spectrin alpha, non-erythrocytic 1; STS: staurosporine; UBE2M: ubiquitin conjugating enzyme E2 M; ZYX: zyxin [ABSTRACT FROM AUTHOR]

The Nomenclature Committee of the International Society of Amyloidosis met at the XVIII International Symposium on Amyloidosis in September and virtually in October 2022 with discussions resulting in this upgraded nomenclature recommendation. The nomenclature principles remain unchanged but there is an ongoing discussion regarding the importance and varying nature of intracellular protein aggregates, particularly those associated with neurodegenerative diseases. Six novel proteins were added to the list of human amyloid fibril proteins. Of these, three are polypeptide hormones and two currently utilised peptide drugs, making the number of known iatrogenic amyloid forms four, all appearing as subcutaneous nodules at the injection site. The sixth novel amyloid fibril protein is the transmembrane 106B protein, forming intracellular amyloid fibrils in disorders associated with frontotemporal dementia. The number of known human amyloid fibril proteins is now 42. [ABSTRACT FROM AUTHOR]

Infertility is a common health problem that affects around 1 in 6 couples in the United States, where half of these cases are attributed to male factors. Genetics play an important role in infertility and it is estimated that up to 50% of cases are due to genetic factors. Despite this, many male infertility cases are still idiopathic. This study aimed to identify the presence of possibly pathogenic rare variants in a set of candidate genes related to azoospermia in 69 Jordanian men using a next-generation sequencing-based panel covering more than a hundred male infertility related genes. A total of 9 variants were found and validated. Among them, two variants included reported pathogenic variants in CFTR and one novel pathogenic variant in the USP9Y gene. We also report the detection of 6 other variants with uncertain significance in other genes. Interestingly, male cases with CFTR variants did not show the expected cystic fibrosis phenotypes except for infertility. This work helps to uncover the contribution of additional genetic factors to the aetiology of male infertility and highlights the importance to obtain more reliable information about the presence of genetic variation in the Jordanian population. [ABSTRACT FROM AUTHOR]

MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions. Abbreviations: BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen's phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature. [ABSTRACT FROM AUTHOR]

Context: As the interest on the research of plant derived bioactive peptides (BPs) for nutraceutical, cosmeceutical and medical applications is increasing, in this work, the application of peptide derived from broccoli to keratinocytes was studied. Objective: We focussed on the characterization of different peptides hydrolysates from broccoli stems [extracted from total protein (E) and from membrane protein (MF)], and their activity when applied to human keratinocytes. Materials and methods: Peptide mixtures from broccoli stems (E and MF) were characterized by proteomics. They were applied to HaCaT cells in order to study cytotoxicity in a concentration range between 20 and 0.15625 mg of protein/mL and wound healing was studied after 24 and 48 h of treatment application. Also, proteomic and gene expression of keratinocytes were analysed. Results: Depending on the source, proteins varied in peptide and amino acid composition. An increased proliferation of keratinocytes was shown after the application of the E peptides mixtures, reaching 190% with the lowest concentrations, but enhanced wound healing repair with E and MF appeared, reaching 59% of wound closure after 48 h. At the gene expression and protein levels of keratinocytes, the upregulation of anti-oncogene p53 and keratinization factors were observed. Discussion: These results suggest that peptide mixtures obtained from broccoli augmented cell proliferation and prevented the carcinogenic, uncontrolled growth of the cells, with different mechanisms depending on the protein source. Conclusions: The results encourage the opening of new lines of research involving the use of Brassica peptides for pharmaceutic or cosmetic use. [ABSTRACT FROM AUTHOR]

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes. Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region. [ABSTRACT FROM AUTHOR]

Mice deficient in GHR (growth hormone receptor; ghr KO) have a dramatic lifespan extension and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in ghr KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice. Abbreviations: CMA: chaperone-mediated autophagy; DIA: data-independent acquisition; ghr KO: growth hormone receptor knockout; GO: gene ontology; I-WAT: inguinal white adipose tissue; KFERQ: a consensus sequence resembling Lys-Phe-Glu-Arg-Gln; LAMP2A: lysosomal-associated membrane protein 2A; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; PBS: phosphate-buffered saline; PI3K: phosphoinositide 3-kinase [ABSTRACT FROM AUTHOR]

The force-from-lipid (FFL) principle states that it is the lateral stretch force from the lipid membrane that ultimately opens mechanosensitive (MS) channels, not the external tether nor the internal cytoskeleton. Piezo channels for certain touch or proprioception and the hair-cell channels for hearing or balance apparently obey this principle, which is based on the idea that the lipid bilayer is an amphipathic compartment with a distinct internal force-distribution profile. Physical stretch or insertion of chemical impurities alters this profile, driving channel shape change to conform to the new environment. Thus, FFL governs all dynamic proteins embedded in membrane, including Kv's and TRPs. This article retraces the humble origin of the FFL concept. Paramecium research first created the mind set and the resources to electrically explore other microbial membranes. Patch clamp revealed MS-channel activities from yeast and E. coli spheroplasts. Despite formidable obstacles against interdisciplinary research, the E. coli MS-channel protein, MscL, was purified through fractionation by following its activity, much like enzyme purification. Reconstituted into a simple lipid bilayer, pure MscL retains mechanosensitivity, thus firmly establishing the FFL principle in 1994. The relatively simple MscL and its functional cousin MscS soon became ideal models for detailed analyses. Like the DNA-RNA-protein 'central dogma' or ATP synthesis, FFL is a fundamental principle, which appeared early in evolution, retained in all cellular life forms, and is expected to contribute to future molecular research on sensations, homeostasis, and embryonic development. [ABSTRACT FROM AUTHOR]

The efficacy of programmed cell death protein 1(PD-1)/Programmed cell death 1 ligand 1 (PD-L1) inhibitors for endometrial cancer remain controversial, and guidelines are inconsistent on which are preferred therapies for advanced disease, or who develop metastases and recurrence. Therefore, we aimed to estimate the efficacy and safety of PD-1/PD-L1 inhibitors in endometrial cancer on a more complete database by adding multiple randomized trials. A systematic and comprehensive search was carried out in PD-1/PD-L1 inhibitors monotherapy. The ORR of PD-1/PDL-1 inhibitors was 29%, and subgroup analysis showed that the pooled ORR of the proficient mismatch repair (pMMR) group was 4% and which was 45% of the deficient mismatch repair (dMMR) group. The DCR of PD-1/PD-L1 inhibitors was 48%, through subgroup analysis, we found that the DCR of the pMMR group was 21% and which was 58% of the dMMR group. The proportion of patients occurring overall adverse events was 65% and grade three or higher adverse events was 14%. The proficient mismatch repair (pMMR) group and the deficient mismatch repair (dMMR) group showed different results. PD-1/PD-L1 inhibitors had shown little success in the pMMR population and better efficacy in the dMMR population. [ABSTRACT FROM AUTHOR]

Integrin αvβ3 is a cell membrane structural protein whose extracellular domain contains a receptor for L-thyroxine (T4). The integrin is expressed in rapidly dividing cells and its internalization is prompted by T4. The protein binds viruses and we have raised the possibility elsewhere that action of free T4 (FT4)—when he latter is increased in the nonthyroidal illness syndrome (NTIS) known to complicate COVID-19 infecction—may enhance cellular uptke of SARS-CoV-2 and its receptor. Because T4 also acts nongenomically via the integrin to promote platelet aggregation and angiogenesis, we suggest here that T4 may contribute to the coagulopathy and endothelial abnormalities that can develop in COVID-19 infections, particularly when the lung is primary affected. Elevated FT4 has been described in the NTIS of COVID-19 patients and may be associated with increased illness severity, but the finding of FT4 elevation is inconsistent in the NTIS literature. Circulating 3,5',3'-triiodo-L-thyronine (reverse T3, rT3) are frequently elevated in NTIS. Thought to be biologically inactive, rT3in fact stimulates cancer cell proliferation via avb3 and also may increase actin polymerization. We propose here that rT3 in the NTIS complicating systemic COVIF-19 infection may support coagulation and disordered blood vessel formation via actin polymerization. [ABSTRACT FROM AUTHOR]

TEX264 (testes expressed gene 264) is a single-pass transmembrane protein, consisting of an N-terminal hydrophobic region, a gyrase inhibitory (GyrI)-like domain, and a loosely structured C terminus. TEX264 was first identified as an endoplasmic reticulum (ER)-resident Atg8-family-binding protein that mediates the degradation of portions of the ER during starvation (i.e., reticulophagy). More recently, TEX264 was identified as a cofactor of VCP/p97 ATPase that promotes the repair of covalently trapped TOP1 (DNA topoisomerase 1)-DNA crosslinks. This review summarizes the current knowledge of TEX264 as a protein with roles in both autophagy and DNA repair and provides an evolutionary and structural analysis of GyrI proteins. Based on our phylogenetic analysis, we provide evidence that TEX264 is a member of a large superfamily of GyrI-like proteins that evolved in bacteria and are present in metazoans, including invertebrates and chordates. Abbreviations: Atg8: autophagy related 8; Atg39: autophagy related 39; Cdc48: cell division cycle 48; CGAS: cyclic GMP-AMP synthase; DPC: DNA-protein crosslinks; DSB: DNA double-strand break; ER: endoplasmic reticulum; GyrI: gyrase inhibitory domain; LRR: leucine-rich repeat; MAFFT: multiple alignment using fast Fourier transform; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; STUBL: SUMO targeted ubiquitin ligase; SUMO: small ubiquitin-like modifier; TEX264: testis expressed gene 264; TOP1cc: topoisomerase 1-cleavage complex; UBZ: ubiquitin binding Zn finger domain; VCP: valosin containing protein [ABSTRACT FROM AUTHOR]

Homologs of qseG gene (coding for the membrane protein QseG), along with the qseEF genes, are present in many Enterobacteriaceae; however, its role in non-pathogenic strains is still unknown. To fill this knowledge gap, we investigated the role of QseG protein of a plant-associated enterobacterium in the interactions with its legume host and in the benefits induced by this enterobacterium in the Mesorhizobium–chickpea symbiosis. Here, we showed that QseG of Kosakonia sp. MH5 is involved in the following processes: (i) the evasion of the plant immune system and (ii) the efficient colonization of chickpea root cells. Furthermore, these features are essential for the beneficial effects of this strain on the Mesorhizobium–chickpea symbiosis. This study demonstrates that the role of QseG is transversal to pathogenic and non-pathogenic enterobacteria and is a step forward to better understanding the molecular bases of plant–bacteria interactions established between legume and beneficial endophytic enterobacteria. [ABSTRACT FROM AUTHOR]

Triptolide (TPL) is a diterpenoid triepoxide with broad antitumor efficacy, while lack of mechanism of action, severe systemic toxicity, and poor water solubility of TPL limited its usage. To unveil the mechanism of action and improve the pharmaceutical properties of TPL, here we explored the molecular mechanism of TPL and then fabricated TPL-loaded membrane protein-chimeric liposomes (TPL@MPLP) and tested its anticancer efficacy against hepatocellular carcinoma (HCC). CCK8 assay, colony formation assay, EdU assay, and flow cytometry were used to examine the activity of TPL. RNA sequence and gain-and-loss of function assays were used to explore the molecular mechanisms. TPL@MP-LP was characterized by size, zeta potential, polydispersity index, and transmission electron microscopy. Cellular uptake and cell viability assay were performed to evaluate the internalization and anticancer efficacy of TPL@MP-LP in vitro. Biodistribution and in vivo antitumor efficacy of TPL@MP-LP were evaluated on orthotopic HCC mice models. TPL robustly inhibited HCC cells by inducing cell proliferation arrest, apoptosis via the mitochondrial pathway, and necroptosis via RIPK1/RIPK3/MLKL signaling. TPL was successfully loaded into MP-LP, with a drug-loading capacity of 5.62 ± 0.80%. MP-LP facilitated TPL internalization and TPL@MP-LP exerted enhanced anticancer efficacy against Huh7 cells. TPL@MP-LP showed targeting ability to the tumor site. More importantly, TPL@MP-LP treatment suppressed tumor growth but showed minimal damage to liver and renal functions. TPL exerted anticancer effects on HCC via inducing cell proliferation arrest, apoptosis, and necroptosis, and the MP-LP might be a promising delivery strategy to improve the antitumor efficacy while mitigating toxicity of TPL for HCC therapy. [ABSTRACT FROM AUTHOR]

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP]3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery. [ABSTRACT FROM AUTHOR]

LAMP1 (lysosomal-associated membrane protein 1) and LAMP2 are the most abundant protein components of lysosome membranes. Both LAMPs have common structures consisting of a large lumenal domain composed of two domains (N-domain and C-domain, which are membrane-distal and -proximal, respectively), both with the β-prism fold, a transmembrane domain, and a short cytoplasmic tail. LAMP2 is involved in various aspects of autophagy, and reportedly forms high-molecular weight complexes at the lysosomal membrane. We previously showed that LAMP2 molecules coimmunoprecipitated with each other, but whether the homophilic interaction is direct or indirect has remained to be elucidated. In the present study, we demonstrated the direct homophilic interaction of mouse LAMP2A molecules, using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces. Specifically, the results suggested that LAMP2A molecules assemble by facing each other with one side of the β-prism (defined as side A) of the C-domains. The N-domain truncation, which increased the coimmunoprecipitation of LAMP2A molecules in our previous study, permitted the nonspecific involvement of both sides of the β-prism (side A and side B). Thus, the presence of the N-domain restricts the LAMP2A interactions to side A-specific. The truncation of LAMP2A impaired the recruitment of GAPDH (a CMA-substrate) fused to the HaloTag protein to the surface of late endosomes/lysosomes (LE/Lys) and affected a process that generates LE/Lys. The present study revealed that the homophilic interaction of LAMP2A is direct, and the side A-specific, homophilic interaction of LAMP2A is required for the functional aspects of LAMP2A. Abbreviations: Aloc-Lys: Nε-allyloxycarbonyl-l-lysine; CMA: chaperone-mediated autophagy; FFE: free-flow electrophoresis; GAPDH-HT: glyceraldehyde-3-phosphate dehydrogenase fused to HaloTag protein; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LBPA: lysobisphosphatidic acid; LE/Lys: late endosome/lysosomes; MEFs: mouse embryonic fibroblasts; pBpa: p-benzoyl- l-phenylalanine [ABSTRACT FROM AUTHOR]

CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis. Abbreviations: AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB [ABSTRACT FROM AUTHOR]

Aberrant chaperone-mediated autophagy (CMA) activation has been suggested as a tumorigenesis-promoting event in various cancers, although its roles in prostate cancer (PCa) remain elusive. Emerging evidence indicates that TPD52 isoform 1, a prostate-specific and androgen-responsive gene, contributes to the malignant progression of PCa. Here, we demonstrate that TPD52 enhances CMA activation by interacting with HSPA8/HSC70 and enhancing substrate degradation in PCa. Elevation of TPD52 is essential for CMA-induced PCa cell proliferation and stress resistance in vitro and in vivo. Furthermore, TPD52 is acetylated by KAT2B at K163, which is a process that can be antagonized by HDAC2. Inactivation of HDAC2 results in elevated TPD52 acetylation, which compromises the interaction between TPD52 and HSPA8, leading to impaired CMA function and tumor growth in vivo. Taken together, our findings reveal that acetylation-dependent regulation of TPD52 modulates CMA oncogenic function in PCa, thereby suggesting the possibility of targeting the TPD52-mediated CMA pathway to control the progression of PCa. Abbreviations: CMA: chaperone-mediated autophagy; HDAC2: histone deacetylase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KAT2B: lysine acetyltransferase 2B; LAMP2A: lysosomal associated membrane protein 2A; PCa: prostate cancer; TPD52: tumor protein D52 [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases. Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type. [ABSTRACT FROM AUTHOR]

Micronuclei are constantly considered as a marker of genome instability and very recently found to be a trigger of innate immune responses. An increased frequency of micronuclei is associated with many diseases, but the mechanism underlying the regulation of micronuclei homeostasis remains largely unknown. Here, we report that CGAS (cyclic GMP-AMP synthase), a known regulator of DNA sensing and DNA repair, reduces the abundance of micronuclei under genotoxic stress in an autophagy-dependent manner. CGAS accumulates in the autophagic machinery and directly interacts with MAP1LC3B/LC3B in a manner dependent upon its MAP1LC3-interacting region (LIR). Importantly, the interaction is essential for MAP1LC3 recruitment to micronuclei and subsequent clearance of micronuclei via autophagy (micronucleophagy) in response to genotoxic stress. Moreover, in contrast to its DNA sensing function to activate micronuclei-driven inflammation, CGAS-mediated micronucleophagy blunts the production of cyclic GMP-AMP (cGAMP) induced by genotoxic stress. We therefore conclude that CGAS is a receptor for the selective autophagic clearance of micronuclei and uncovered an unprecedented role of CGAS in micronuclei homeostasis to dampen innate immune surveillance. Abbreviations: ATG: autophagy-related; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LIR, MAP1LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NDZ: nocodazole; STING1: stimulator of interferon response cGAMP interactor 1 [ABSTRACT FROM AUTHOR]

Coxsackievirus B3 (CVB3) is a prevalent etiological agent for viral myocarditis and neurological disorders, particularly in infants and young children. Virus-encoded proteinases have emerged as cytopathic factors that contribute to disease pathogenesis in part through targeting the cellular recycling machinery of autophagy. Although it is appreciated that CVB3 can usurp cellular macroautophagy/autophagy for pro-viral functions, the precise mechanisms by which viral proteinases disrupt autophagy remain incompletely understood. Here we identified TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, as a novel target of CVB3 proteinase 3 C. Time-course infections uncovered a significant loss of full-length TFEB and the emergence of a lower-molecular mass (~63 kDa) fragment. Cellular and in vitro cleavage assays revealed the involvement of viral proteinase 3 C in the proteolytic processing of TFEB, while site-directed mutagenesis identified the site of cleavage after glutamine 60. Assessment of TFEB transcriptional activity using a reporter construct discovered a loss of function of the cleavage fragment despite nuclear localization and retaining of the ability of DNA and protein binding. Furthermore, we showed that CVB3 infection was also able to trigger cleavage-independent nuclear translocation of TFEB that relied on the serine-threonine phosphatase PPP3/calcineurin. Finally, we demonstrated that both TFEB and TFEB [Δ60] serve roles in viral egress albeit through differing mechanisms. Collectively, this study reveals that CVB3 targets TFEB for proteolytic processing to disrupt host lysosomal function and enhance viral infection. Abbreviations:ACTB: actin beta; CLEAR: coordinated lysosomal enhancement and regulation; CVB3: coxsackievirus B3; DAPI: 4′,6-diamidino-2-phenylindole; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LTR: LysoTracker Red; PPP3/calcineurin: protein phosphatase 3; PPP3CA: protein phosphatase 3 catalytic subunit A; p-TFEB: phospho-Ser211 TFEB; si-CON: scramble control siRNA; TFEB: transcription factor EB; TFEB [Δ60]: TFEB cleavage fragment that lacks the first 60 amino acids; VP1: viral capsid protein 1 [ABSTRACT FROM AUTHOR]

In this study, clinical and biochemical methods were utilized to predict the final diagnosis of hereditary spherocytosis (HS), correlate the diagnosis with splenectomy, and examine the usefulness of this approach. We biochemically and cytochemically analysed erythrocyte membrane proteins before making a final HS diagnosis based on gene analysis to compare diagnostic approaches. The clinical features of six patients with various subtypes of HS and symptoms were observed by blood analysis using eosin-5′-maleimide staining, biochemical analysis using sodium dodecyl sulphate – polyacrylamide gel electrophoresis with western blotting, and mass spectrometry. Finally, diagnostic membrane gene analysis was performed. Five of the six patients showed mild to moderate or severe anaemia, and the other patient was non-anaemic; all six patients showed faint eosin-5′-maleimide staining. In western blotting of erythrocyte membrane proteins, all six patients (three with β-spectrin, two with ankyrin, and one with SLC4A1 anomalies) showed low-molecular-weight peptide fragments, which were confirmed by mass spectrometry in the region corresponding to the band 3 protein. The two patients with an ankyrin gene anomaly exhibited severe anaemia, and two patients with simultaneous SLC4A1, SPTB, and UGT1A1 anomalies exhibited mild anaemia and hyperbilirubinemia. We determined the relationship among clinical features, cytochemical parameters, and gene anomalies in six patients with newly diagnosed HS while referring to previously published cases. These findings reveal a close relationship between clinical features and membrane characteristics in HS, which can facilitate diagnosis and inform treatment. [ABSTRACT FROM AUTHOR]

The autophagy-lysosome system is an important cellular degradation pathway that recycles dysfunctional organelles and cytotoxic protein aggregates. A decline in this system is pathogenic in many human diseases including neurodegenerative disorders, fatty liver disease, and atherosclerosis. Thus there is intense interest in discovering therapeutics aimed at stimulating the autophagy-lysosome system. Trehalose is a natural disaccharide composed of two glucose molecules linked by a ɑ-1,1-glycosidic bond with the unique ability to induce cellular macroautophagy/autophagy and with reported efficacy on mitigating several diseases where autophagy is dysfunctional. Interestingly, the mechanism by which trehalose induces autophagy is unknown. One suggested mechanism is its ability to activate TFEB (transcription factor EB), the master transcriptional regulator of autophagy-lysosomal biogenesis. Here we describe a potential mechanism involving direct trehalose action on the lysosome. We find trehalose is endocytically taken up by cells and accumulates within the endolysosomal system. This leads to a low-grade lysosomal stress with mild elevation of lysosomal pH, which acts as a potent stimulus for TFEB activation and nuclear translocation. This process appears to involve inactivation of MTORC1, a known negative regulator of TFEB which is sensitive to perturbations in lysosomal pH. Taken together, our data show the trehalose can act as a weak inhibitor of the lysosome which serves as a trigger for TFEB activation. Our work not only sheds light on trehalose action but suggests that mild alternation of lysosomal pH can be a novel method of inducing the autophagy-lysosome system. Abbreviations: ASO: antisense oligonucleotide; AU: arbitrary units; BMDM: bone marrow-derived macrophages; CLFs: crude lysosomal fractions; CTSD: cathepsin D; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; MAP1LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; pMAC: peritoneal macrophages; SLC2A8/GLUT8: solute carrier family 2, (facilitated glucose transporter), member 8; TFEB: transcription factor EB; TMR: tetramethylrhodamine; TREH: trehalase [ABSTRACT FROM AUTHOR]

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12–ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation. Abbreviations: CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1. [ABSTRACT FROM AUTHOR]

The mechanisms orchestrating recycling of lysosomes through autophagic lysosome reformation (ALR) is incompletely understood. Previous data show that genetic depletion of BLOC1S1/GCN5L1/BORCS1 increases autolysosome (AL) accumulation. We postulated that this phenotype may manifest due to perturbed ALR. We explored this in control and bloc1s1 liver-specific knockout (LKO) mouse hepatocytes, showing that in response to nutrient-deprivation LKO's fail to initiate ALR due to blunted lysosomal tubulation. As kinesin motor proteins and the intracellular cytoskeleton are requirements for tubular formation from ALs, we explored the interaction of BLOC1S1 with motor proteins and cytoskeletal factors. BLOC1S1 interacts with the ARL8B-KIF5B (GTPase and kinesin motor protein) complex to recruit KIF5B to ALs. Furthermore, BLOC1S1 interacts with the actin nucleation promoting factor WHAMM, which is an essential structural protein in the initiation of lysosomal tubulation (LT). Interestingly, the genetic reintroduction of BLOC1S1 rescues LT in LKO hepatocytes, but not when KIF5B is concurrently depleted. Finally, given the central role of MTORC1 signaling in ALR initiation, it was interesting that MTORC1 activity was increased despite the absence of LT in LKO hepatocytes. Concurrently, inhibition of MTORC1 abolished BLOC1S1 reconstitution-mediated rescue of LT in LKO hepatocytes. Taken together these data demonstrate that the functional interaction of BLOC1S1 with the kinesin binding complex and the actin cytoskeleton are a requirement for LT which, in parallel with MTORC1 signaling, initiate lysosome recycling via ALR. Abbreviations: 3-MA: 3-methyladenine; AL: autolysosome; ALR: autophagic lysosome reformation; ARL8B: ADP-ribosylation factor-like protein 8B; ARPC2: actin related protein 2/3 complex, subunit 2; ATAT1/αTAT1: alpha tubulin acetyltransferase 1; AVd: autophagic vacuoles, degradative; BLOC1S1/GCN5L1: biogenesis of lysosomal organelles complex-1, subunit 1; CQ: chloroquine; KIF5B: kinesin family member 5B; KLC1: kinesin light chain 1; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC3B-I: cytosolic form of LC3B; LC3B-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; LKO: liver-specific knockout; LIs: lysosome inhibitors; LT: lysosomal tubulation; Ly: lysosome; MTORC1: mechanistic target of rapamycin kinase complex 1; PLEKHM2/SKIP: pleckstrin homology domain containing, family M (with RUN domain) member 2; Snapin: SNAP-associated protein; SQSTM1/p62: sequestosome 1; SVPs: synaptic vesicle precursors; TFEB: transcription Factor EB; TFE3: transcription factor E3; WHAMM: WAS protein homolog associated with actin, golgi membranes and microtubules. [ABSTRACT FROM AUTHOR]

In humans, TDRD7 (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7's functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in tdrd7-deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to Tbc1d20 mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that tdrd7 deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human. Abbreviations: CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle's balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type [ABSTRACT FROM AUTHOR]

Platelet Endothelial Aggregation Receptor 1 (PEAR1) is an orphan receptor of unknown function which mediates powerful activation of platelets and endothelial cells in response to crosslinking by antibodies and sulfated polysaccharides belonging to the dextran and fucoidan families. PEAR1 is a single transmembrane protein composed of 15 epidermal growth factor-like repeat sequences and with a conserved binding motif, YXXM, which when phosphorylated binds to phosphoinositide 3-kinase (PI3K). The 13th of the repeats has a heparin-binding sequence that is the site of interaction with the sulfated fucoidans and the only known endogenous ligand FcεRIα. Crosslinking of PEAR1 drives Src family kinase phosphorylation of the cytosolic tail leading to binding and activation of PI3K. In this Opinion Article, we summarize the literature on PEAR1 expression, structure and signaling, and the search for further endogenous ligands. We highlight one article in which phosphorylation of a 150 kDa platelet protein by heparin-containing ligands has been reported and propose that PEAR1 is a receptor for one or more glycosaminoglycan-conjugated proteins (proteoglycans). The up-regulation of PEAR1 at sites of inflammation in the vasculature and its role in angiogenesis suggests a role in the interplay of inflammation, platelets, coagulation, and thromboinflammation. We speculate that this may explain the link between single nucleotide variants in PEAR1 and cardiovascular disease. [ABSTRACT FROM AUTHOR]

Our previous studies reveal a mechanism for lipid droplet (LD)-mediated proteostasis in the endoplasmic reticulum (ER) whereby unfolded proteins that accumulate in the ER in response to lipid imbalance-induced ER stress are removed by LDs and degraded by microlipophagy (µLP), autophagosome-independent LD uptake into the vacuole (the yeast lysosome). Here, we show that dithiothreitol- or tunicamycin-induced ER stress also induces µLP and identify an unexpected role for vacuolar membrane dynamics in this process. All stressors studied induce vacuolar fragmentation prior to µLP. Moreover, during µLP, fragmented vacuoles fuse to form cup-shaped structures that encapsulate and ultimately take up LDs. Our studies also indicate that proteins of the endosome sorting complexes required for transport (ESCRT) are upregulated, required for µLP, and recruited to LDs, vacuolar membranes, and sites of vacuolar membrane scission during µLP. We identify possible target proteins for LD-mediated ER proteostasis. Our live-cell imaging studies reveal that one potential target (Nup159) localizes to punctate structures that colocalizes with LDs 1) during movement from ER membranes to the cytosol, 2) during microautophagic uptake into vacuoles, and 3) within the vacuolar lumen. Finally, we find that mutations that inhibit LD biogenesis, homotypic vacuolar membrane fusion or ESCRT function inhibit stress-induced autophagy of Nup159 and other ER proteins. Thus, we have obtained the first direct evidence that LDs and µLP can mediate ER stress-induced ER proteostasis, and identified direct roles for ESCRT and vacuolar membrane fusion in that process. [ABSTRACT FROM AUTHOR]

Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux. Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type. [ABSTRACT FROM AUTHOR]