SEC23B Loss-of-Function Suppresses Hepcidin Expression by Impairing Glycosylation Pathway in Human Hepatic Cells.

Biallelic pathogenic variants in the SEC23B gene cause congenital dyserythropoietic anemia type II (CDA II), a rare hereditary disorder hallmarked by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypo-glycosylation of some red blood cell membrane proteins. Abnormalities in SEC23B, which encodes the homonymous cytoplasmic COPII (coat protein complex II) component, disturb the endoplasmic reticulum to Golgi trafficking and affect different glycosylation pathways. The most harmful complication of CDA II is the severe iron overload. Within our case series (28 CDA II patients), approximately 36% of them exhibit severe iron overload despite mild degree of anemia and slightly increased levels of ERFE (the only erythroid regulator of hepcidin suppression). Thus, we hypothesized a direct role of SEC23B loss-of-function in the pathomechanism of hepatic iron overload. We established a hepatic cell line, HuH7, stably silenced for SEC23B. In silenced cells, we observed significant alterations of the iron status, due to both the alteration in BMP/SMADs pathway effectors and a reduced capability to sense BMP6 stimulus. We demonstrated that the loss-of-function of SEC23B is responsible of the impairment in glycosylation of the membrane proteins involved in the activation of the BMP/SMADs pathway with subsequent hepcidin suppression. Most of these data were confirmed in another hepatic cell line, HepG2, stably silenced for SEC23B. Our findings suggested that the pathogenic mechanism of iron overload in CDA II is associated to both ineffective erythropoiesis and to a specific involvement of SEC23B pathogenic variants at hepatic level. Finally, we demonstrated the ability of SEC23B paralog, i.e., SEC23A, to rescue the hepcidin suppression, highlighting the functional overlap between the two SEC23 paralogs in human hepatic cells.

Alzheimer's vulnerable brain region relies on a distinct retromer core dedicated to endosomal recycling.

Whether and how the pathogenic disruptions in endosomal trafficking observed in Alzheimer's disease (AD) are linked to its anatomical vulnerability remain unknown. Here, we began addressing these questions by showing that neurons are enriched with a second retromer core, organized around VPS26b, differentially dedicated to endosomal recycling. Next, by imaging mouse models, we show that the trans-entorhinal cortex, a region most vulnerable to AD, is most susceptible to VPS26b depletion-a finding validated by electrophysiology, immunocytochemistry, and behavior. VPS26b was then found enriched in the trans-entorhinal cortex of human brains, where both VPS26b and the retromer-related receptor SORL1 were found deficient in AD. Finally, by regulating glutamate receptor and SORL1 recycling, we show that VPS26b can mediate regionally selective synaptic dysfunction and SORL1 deficiency. Together with the trans-entorhinal's unique network properties, hypothesized to impose a heavy demand on endosomal recycling, these results suggest a general mechanism that can explain AD's regional vulnerability.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition.

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson's disease. Here, we study the basic neurobiology of VPS35 and Parkinson's disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

TANGO1 marshals the early secretory pathway for cargo export.

TANGO1 protein facilitates the endoplasmic reticulum (ER) export of large cargoes that cannot be accommodated in 60 nm transport vesicles. It assembles into a ring in the plane of the ER membrane to create a distinct domain. Its lumenal portion collects and sorts folded cargoes while its cytoplasmic domains collar COPII coats, recruit retrograde COPI-coated membranes that fuse within the TANGO1 ring, thus opening a tunnel for cargo transfer from the ER into a growing export conduit. This mode of cargo transfer bypasses the need for vesicular intermediates and is used to export the most abundant and bulky cargoes. The evolution of TANGO1 and its activities defines the difference between yeast and animal early secretory pathways.

Small Rab GTPases in Intracellular Vesicle Trafficking: The Case of Rab3A/Raphillin-3A Complex in the Kidney.

Small Rab GTPases, the largest group of small monomeric GTPases, regulate vesicle trafficking in cells, which are integral to many cellular processes. Their role in neurological diseases, such as cancer and inflammation have been extensively studied, but their implication in kidney disease has not been researched in depth. Rab3a and its effector Rabphillin-3A (Rph3A) expression have been demonstrated to be present in the podocytes of normal kidneys of mice rats and humans, around vesicles contained in the foot processes, and they are overexpressed in diseases with proteinuria. In addition, the Rab3A knockout mice model induced profound cytoskeletal changes in podocytes of high glucose fed animals. Likewise, RphA interference in the Drosophila model produced structural and functional damage in nephrocytes with reduction in filtration capacities and nephrocyte number. Changes in the structure of cardiac fiber in the same RphA-interference model, open the question if Rab3A dysfunction would produce simultaneous damage in the heart and kidney cells, an attractive field that will require attention in the future.

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function.

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.

TBC1D8B, a GTPase-activating protein, is a novel apoptosis inducer.

Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.

The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in our forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.

Ca2+ sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons.

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.

Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins.

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.

Disrupted glucose homeostasis and skeletal-muscle-specific glucose uptake in an exocyst knockout mouse model.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.

TRIM72 mediates lung epithelial cell death upon hyperoxia exposure.

BACKGROUND: Premature infants often require oxygen (O2) therapy for respiratory distress syndrome; however, excessive use of O2 can cause clinical conditions such as bronchopulmonary dysplasia. Although many treatment methods are currently available, they are not effective in preventing bronchopulmonary dysplasia. Herein, we explored the role of tripartite motif protein 72 (TRIM72), a factor involved in repairing alveolar epithelial wounds, in regulating alveolar cells upon hyperoxia exposure. METHODS: In this in vivo study, we used Sprague-Dawley rat pups that were reared in room air or 85% O2 for 2 weeks after birth. The lungs were excised for histological analyses, and TRIM72 expression was assessed on postnatal days 7 and 14. For in vitro experiments, RLE-6TN cells (i.e., rat alveolar type II epithelial cells) and A549 cells (i.e., human lung carcinoma epithelial cells) were exposed to 85% O2 for 5 days. The cells were then analyzed for cell viability, and TRIM72 expression was determined. RESULTS: Exposure to hyperoxia reduced body and lung weight, increased mean linear intercept values, and upregulated TRIM72 expression. In vitro study results revealed increased or decreased lung cell viability upon hyperoxia exposure depending on the suppression or overexpression of TRIM72, respectively. CONCLUSION: Hyperoxia upregulates TRIM72 expression in neonatal rat lung tissue; moreover, it initiates TRIM72-dependent alveolar epithelial cell death, leading to hyperoxia-induced lung injury.

Vac8 determines phagophore assembly site vacuolar localization during nitrogen starvation-induced autophagy.

Macroautophagy/autophagy is a key catabolic process in which different cellular components are sequestered inside double-membrane vesicles called autophagosomes for subsequent degradation. In yeast, autophagosome formation occurs at the phagophore assembly site (PAS), a specific perivacuolar location that works as an organizing center for the recruitment of different autophagy-related (Atg) proteins. How the PAS is localized to the vacuolar periphery is not well understood. Here we show that the vacuolar membrane protein Vac8 is required for correct vacuolar localization of the PAS. We provide evidence that Vac8 anchors the PAS to the vacuolar membrane by binding Atg13 and recruiting the Atg1 initiation complex. VAC8 deletion or mislocalization of the protein reduce autophagy activity, highlighting the importance of both the PAS and the correct vacuolar localization of the Atg1 initiation complex for efficient and robust autophagy.Abbreviations: AID: auxin-inducible degradation; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; GFP: green fluorescent protein; IAA: 3-indole acetic acid; PAS: phagophore assembly site; RFP: red fluorescent protein.

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis.

In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.

An ER-Golgi Tethering Factor SLOH4/MIP3 Is Involved in Long-Term Heat Tolerance of Arabidopsis.

Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L-heat stress but not to S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2 and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1-1 mutants was similar to that of the wild type (WT). Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the WT. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1) - accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins - were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not by bZIP28, resulting in the initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in the MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER-Golgi vesicle tethering.

Human VAPome Analysis Reveals MOSPD1 and MOSPD3 as Membrane Contact Site Proteins Interacting with FFAT-Related FFNT Motifs.

Membrane contact sites (MCS) are intracellular regions where two organelles come closer to exchange information and material. The majority of the endoplasmic reticulum (ER) MCS are attributed to the ER-localized tether proteins VAPA, VAPB, and MOSPD2. These recruit other proteins to the ER by interacting with their FFAT motifs. Here, we describe MOSPD1 and MOSPD3 as ER-localized tethers interacting with FFAT motif-containing proteins. Using BioID, we identify proteins interacting with VAP and MOSPD proteins and find that MOSPD1 and MOSPD3 prefer unconventional FFAT-related FFNT (two phenylalanines [FF] in a neutral tract) motifs. Moreover, VAPA/VAPB/MOSPD2 and MOSPD1/MOSPD3 assemble into two separate ER-resident complexes to interact with FFAT and FFNT motifs, respectively. Because of their ability to interact with FFNT motifs, MOSPD1 and MOSPD3 could form MCS between the ER and other organelles. Collectively, these findings expand the VAP family of proteins and highlight two separate complexes in control of interactions between intracellular compartments.

TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes.

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

S100A8 transported by SEC23A inhibits metastatic colonization via autocrine activation of autophagy.

Metastasis is the main cause of failure of cancer treatment. Metastatic colonization is regarded the most rate-limiting step of metastasis and is subjected to regulation by a plethora of biological factors and processes. On one hand, regulation of metastatic colonization by autophagy appears to be stage- and context-dependent, whereas mechanistic characterization remains elusive. On the other hand, interactions between the tumor cells and their microenvironment in metastasis have long been appreciated, whether the secretome of tumor cells can effectively reshape the tumor microenvironment has not been elucidated mechanistically. In the present study, we have identified "SEC23A-S1008-BECLIN1-autophagy axis" in the autophagic regulation of metastatic colonization step, a mechanism that tumor cells can exploit autophagy to exert self-restrain for clonogenic proliferation before the favorable tumor microenvironment is established. Specifically, we employed a paired lung-derived oligometastatic cell line (OL) and the homologous polymetastatic cell line (POL) from human melanoma cell line M14 that differ in colonization efficiency. We show that S100A8 transported by SEC23A inhibits metastatic colonization via autocrine activation of autophagy. Furthermore, we verified the clinical relevance of our experimental findings by bioinformatics analysis of the expression of Sec23a and S100A8 and the clinical-pathological associations. We demonstrate that higher Sec23a and Atg5 expression levels appear to be protective factors and favorable diagnostic (TNM staging) and prognostic (overall survival) markers for skin cutaneous melanoma (SKCM) and colon adenocarcinoma (COAD) patients. And we confirm the bioinformatics analysis results with SKCM biopsy samples.

Scaffold proteins in bulk and selective autophagy.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases.