Diverse Fgfr1 signaling pathways and endocytic trafficking regulate mesoderm development.

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1-null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Dynein functions in galectin-3 mediated processes of clathrin-independent endocytosis.

Multiple endocytic processes operate in cells in tandem to uptake multiple cargoes involved in diverse cellular functions, including cell adhesion and migration. The best-studied clathrin-mediated endocytosis (CME) involves the formation of a well-defined cytoplasmic clathrin coat to facilitate cargo uptake. According to the glycolipid-lectin (GL-Lect) hypothesis, galectin-3 (Gal3) binds to glycosylated membrane receptors and glycosphingolipids (GSLs) to drive membrane bending and tubular membrane invaginations that undergo scission to form a morphologically distinct class of uptake structures, termed clathrin-independent carriers (CLICs). Which components from cytoskeletal machinery are involved in the scission of CLICs remains to be explored. In this study, we propose that dynein is recruited onto Gal3-induced tubular endocytic pits and provides the pulling force for friction-driven scission. The uptake of Gal3 and its cargoes (CD98/CD147) is significantly dependent on dynein activity, whereas only transferrin (CME marker) is slightly affected upon dynein inhibition. Our study reveals that Gal3 and Gal3-dependent (CD98 and CD147) clathrin-independent cargoes require dynein for the clathrin-independent endocytosis.

The CHD family chromatin remodeling enzyme, Kismet, promotes both clathrin-mediated and activity-dependent bulk endocytosis.

Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3ß expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.

Aagab is required for zebrafish larval development by regulating neural activity.

Clathrin-mediated endocytosis has been implicated in various physiological processes, including nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation. Despite prior knowledge of its importance as a key regulator in promoting clathrin-mediated endocytosis, the physiological function of α- and γ-adaptin binding protein (aagab) remains elusive. In this study, we investigate the biological function of aagab during zebrafish development. We establish a loss-of-function mutant of aagab in zebrafish, revealing impaired swimming and early larval mortality. Given the high expression level of aagab in the brain, we probe into its physiological role in the nervous system. aagab mutants display subdued calcium responses and local field potential in the optic tectal neurons, aligning with reduced neurotransmitter release (e.g., norepinephrine) in the tectal neuropil of aagab mutants. Overexpressing aagab mRNA or nervous stimulant treatment in mutants restores neurotransmitter release, calcium responses, swimming ability, and survival. Furthermore, our observations show delayed release of FM 1-43 in AAGAB knockdown differentiated neuroblastoma cells, pointing towards a probable link to defective clathrin-mediated synaptic vesicle recycling. In conclusion, our study underscores the significance of Aagab in neurobiology and suggests its potential impacts on neurological disorders.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Targeting of insulin receptor endocytosis as a treatment to insulin resistance.

BACKGROUND: Insulin resistance is the decreased effectiveness of insulin receptor function during signaling of glucose uptake. Insulin receptors are regulated by endocytosis, a process that removes receptors from the cell surface to be marked for degradation or for re-use. OBJECTIVES: Our goal was to discover insulin-resistance-related genes that play key roles in endocytosis which could serve as potential biological targets to enhance insulin sensitivity. METHODS: The gene mutations related to insulin resistance were elucidated from ClinVar. These were used as the seed set. Using the GeneFriends program, the genes associated with this set were elucidated and used as an enriched set for the next step. The enriched gene set network was visualized by Cytoscape. After that, using the VisANT program, the most significant cluster of genes was identified. With the help of the DAVID program, the most important KEGG pathway corresponding to the gene cluster and insulin resistance was found. Eleven genes part of the KEGG endocytosis pathway were identified. Finally, using the ChEA3 program, seven transcription factors managing these genes were defined. RESULTS: Thirty-two genes of pathogenic significance in insulin resistance were elucidated, and then co-expression data for these genes were utilized. These genes were organized into clusters, one of which was singled out for its high node count of 58 genes and low p-value (p = 4.117 × 10-7). DAVID Pathways, a functional annotation tool, helped identify a set of 11 genes from a single cluster associated with the endocytosis pathway related to insulin resistance. These genes (AMPH, BIN1, CBL, DNM1, DNM2, DNM3, ITCH, SH3GL1, SH3GL2, SH3GL3, and SH3KBP1) are all involved in either clathrin-mediated endocytosis of the insulin receptor (IR) or clathrin-independent endocytosis of insulin-resistance-related G protein-coupled receptors (GPCR). They represent prime therapeutic targets to improve insulin sensitivity through modulation of transmembrane cell signaling. Using the ChEA3 database, we also found seven transcription factors (REST, MYPOP, CAMTA2, MYT1L, ZBTB18, NKX6-2, and CXXC5) that control the expression of these 11 genes. Inhibiting these key transcription factors would be another strategy to downregulate endocytosis. CONCLUSION: We believe that delaying removal of insulin receptors from the cell surface would prolong signaling of glucose uptake and counteract the symptoms of insulin resistance.

Endocytosis and Endocytic Motifs across the Connexin Gene Family.

Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.

Phosphorylation of ADAPTOR PROTEIN-2 µ-adaptin by ADAPTOR-ASSOCIATED KINASE1 regulates the tropic growth of Arabidopsis roots.

ADAPTOR-ASSOCIATED PROTEIN KINASE1 (AAK1) is a known regulator of clathrin-mediated endocytosis in mammals. Human AAK1 phosphorylates the µ2 subunit of the ADAPTOR PROTEIN-2 (AP-2) complex (AP2M) and plays important roles in cell differentiation and development. Previous interactome studies discovered the association of AAK1 with AP-2 in Arabidopsis (Arabidopsis thaliana), but its function was unclear. Here, genetic analysis revealed that the Arabidopsis aak1 and ap2m mutants both displayed altered root tropic growth, including impaired touch- and gravity-sensing responses. In Arabidopsis, AAK1-phosphorylated AP2M on Thr-163, and expression of the phospho-null version of AP2M in the ap2m mutant led to an aak1-like phenotype, whereas the phospho-mimic forms of AP2M rescued the aak1 mutant. In addition, we found that the AAK1-dependent phosphorylation state of AP2M modulates the frequency distribution of endocytosis. Our data indicate that the phosphorylation of AP2M on Thr-163 by AAK1 fine-tunes endocytosis in the Arabidopsis root to control its tropic growth.

A Putative Zn(II)2Cys6-Type Transcription Factor FpUme18 Is Required for Development, Conidiation, Cell Wall Integrity, Endocytosis and Full Virulence in Fusarium pseudograminearum.

Fusarium pseudograminearum is one of the major fungal pathogens that cause Fusarium crown rot (FCR) worldwide and can lead to a substantially reduced grain yield and quality. Transcription factors play an important role in regulating growth and pathogenicity in plant pathogens. In this study, we identified a putative Zn(II)2Cys6 fungal-type domain-containing transcription factor and named it FpUme18. The expression of FpUME18 was induced during the infection of wheat by F. pseudograminearum. The ΔFpume18 deletion mutant showed defects in growth, conidial production, and conidial germination. In the responses to the cell wall, salt and oxidative stresses, the ΔFpume18 mutant inhibited the rate of mycelial growth at a higher rate compared with the wild type. The staining of conidia and mycelia with lipophilic dye FM4-64 revealed a delay in endocytosis when FpUME18 was deleted. FpUME18 also positively regulated the expression of phospholipid-related synthesis genes. The deletion of FpUME18 attenuated the pathogenicity of wheat coleoptiles. FpUME18 also participated in the production of the DON toxin by regulating the expression of TRI genes. Collectively, FpUme18 is required for vegetative growth, conidiation, stress response, endocytosis, and full virulence in F. pseudograminearum.

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles.

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid-based therapeutics transported by nanoparticles. Utilizing a siRNA-targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3-LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID). A cell-based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data-rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid-phase endocytosis as a productive cellular entry route. With this new knowledge, MC3-LNP is re-engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE-ID approach can be broadly applicable for optimizing nanomedicine-based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid-based therapeutics.

Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.

HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis.

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.

Regulation of transferrin receptor trafficking by optineurin and its disease-associated mutants.

Transferrin receptor (TFRC) is a transmembrane protein that plays a crucial role in mediating homeostasis of iron in the cell. The binding of transferrin (that is bound to iron) to TFRC at the cell membrane generally starts endocytosis of TFRC-transferrin complex, which leads to formation of vesicles that are positive for TFRC. These vesicles travel to the early endosomes and later to the endocytic recycling compartment. Release of iron occurs in the early endosomes because of acidic pH. Major fraction of the transferrin and TFRC is transported back to the cell membrane; however, a minor fraction of it is transported to lysosomes through the process of autophagy. Optineurin (OPTN) is a multi-functional adaptor protein that plays a pivotal role in the control of TFRC trafficking, recycling and autophagy dependent degradation. Optineurin also plays a role in cargo-selective and non-selective autophagy. Here, we review our understanding of the function of OPTN in regulating TFRC trafficking, recycling and autophagy dependent degradation. We also discuss the mechanisms by which certain disease-associated mutations of OPTN alter these processes.

SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes.

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagellar pocket was not affected by knockdown of NRP1. However the quantity of Tf per endosome dropped significantly, consistent with NRP1 promoting robust capture and/or retention of Tf in vesicles. NRP1 is involved in motility of Tf-laden vesicles since distances between endosomes and the kinetoplast were reduced after knockdown of the gene. In search of possible mediators of NRP1 modulation of Tf endocytosis, the gene was knocked down and the phosphoproteome analyzed. Phosphorylation of protein kinases forkhead, NEK6, and MAPK10 was altered, in addition to EpsinR, synaptobrevin and other vesicle-associated proteins predicted to be involved in endocytosis. These candidate proteins may link NRP1 functionally either to protein kinases or to vesicle-associated proteins.

Endocytosis of Peptidase Inhibitor SerpinE2 promotes Myocardial Fibrosis through activating ERK1/2 and ß-catenin Signaling Pathways.

Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and ß-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, ß-catenin signaling pathways, consequently promoting collagen production.