Cargo Proteins Research Articles

Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

Characterization of the Secretome from Spheroids of Adipose-Derived Stem Cells (SASCs) and Its Potential for Tissue Regeneration.

Spheroids are spherical aggregates of cells that mimic the three-dimensional (3D) architecture of tissues more closely than traditional two dimensional (2D) cultures. Spheroids of adipose stem cells (SASCs) show special features such as high multilineage differentiation potential and immunomodulatory activity. These properties have been attributed to their secreted factors, such as cytokines and growth factors. Moreover, a key role is played by the extracellular vesicles (EVs), which lead a heterogeneous cargo of proteins, mRNAs, and small RNAs that interfere with the pathways of the recipient cells. The aim of this work was to characterize the composition of the secretome and exosome from SASCs and evaluate their regenerative potential. SASCs were extracted from adipose samples of healthy individuals after signing informed consent. The exosomes were isolated and characterized by Dinamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western blotting analyses. The expression of mRNAs and miRNAs were evaluated through real-time PCR. Lastly, a wound-healing assay was performed to investigate their regenerative potential on different cell cultures. The SASCs' exosomes showed an up-regulation of NANOG and SOX2 mRNAs, typical of stemness maintenance, as well as miR126 and miR146a, related to angiogenic and osteogenic processes. Moreover, the exosomes showed a regenerative effect. The SASCs' secretome carried paracrine signals involved in stemness maintenance, pro-angiogenic and pro-osteogenic differentiation, immune system regulation, and regeneration.

Oncoviral Infections and Small Extracellular Vesicles.

Small extracellular vesicles (sEV) are small membrane-bound nanovesicles with a size range below 200 nm that are released by all types of cells. sEV carry a diverse cargo of proteins, lipids, glycans, and nucleic acids that mimic the content of producer cells. sEV mediate intercellular communication and play a key role in a broad variety of physiological and pathological conditions. Recently, numerous reports have emerged examining the role of sEV in viral infections. A significant number of similarities in the sEV biogenesis pathways and the replication cycles of viruses suggest that sEV might influence the course of viral infections in diverse ways. Besides directly modulating virus propagation by transporting the viral cargo (complete virions, proteins, RNA, and DNA), sEV can also modify the host antiviral response and increase the susceptibility of cells to infection. The network of mutual interactions is particularly complex in the case of oncogenic viruses, deserving special consideration because of its significance in cancer progression. This review summarizes the current knowledge of interactions between sEV and oncogenic viruses, focusing on sEV abilities to modulate the carcinogenic properties of oncoviruses.

The endolysosomal system in conventional and unconventional protein secretion.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities.

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

Lysosomal Activation Mediated by Endocytosis in J774 Cell Culture Treated with N-Trimethyl Chitosan Nanoparticles.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 μg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Structural Investigations of Cargo Molecules Inside Icosahedrally Symmetric Encapsulin by VUVCD Spectroscopic Measurements.

Prokaryotes organize intracellular compartments with protein-based organelles called encapsulins. Encapsulins with icosahedral symmetry can encapsulate specific cargo proteins mediated by targeting peptides or encapsulation-mediating domains. Encapsulins have been used in eukaryotic cells for bioengineering, vaccine development, and nanoparticle alignment. Their versatility makes them attractive for research; however, detailed structural information on encapsulins is crucial for further applied research. However, cargo proteins are randomly oriented inside the icosahedral encapsulins. The random orientation of cargo proteins presents a challenge for structural analysis that relies on averaging processes such as x-ray crystallography and cryo-electron microscopy (cryo-EM) single-particle imaging. Therefore, we aimed to accurately estimate the secondary structure content and elucidate the structure of cargo proteins inside the particle by measuring the circular dichroism (CD) spectra using vacuum ultraviolet circular dichroism (VUVCD) spectroscopy. Thus, the structure of the cargo protein inside encapsulin was evaluated. This approach could potentially set a standard for evaluating cargo proteins inside particles in future applied research on encapsulins.

Establishing the capacity to monitor proteins relevant to the study of drug exposure and response using liver-derived extracellular vesicles.

Drug exposure and response is determined by pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Interindividual differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin PK and PD variability and impact treatment efficacy and tolerability. Extracellular vesicles (EVs) carry protein cargo inherited from originating cells and may be useful for defining differences in key proteins related to hepatic drug metabolism and the treatment of metabolic-associated fatty liver disease (MAFLD). We sought to quantify these proteins in liver-derived EVs and establish the profile relative to paired tissue. EVs were recovered from human liver tissue samples (LT-EV, n = 11). Targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays were employed for absolute quantification of proteins in EV isolates and matched liver tissue. DMEs and MAFLD drug targets were readily detected and quantified in LT-EVs. Twelve of 15 DMEs exhibited moderate to strong correlation (Spearman ⍴ = 0.618-0.973) between tissue and EVs. Correlation in protein abundance was influenced by the extent of extra-hepatic expression of the target. This study provides evidence that key proteins related to PK and PD profiles can be measured in liver-derived EVs and abundance of liver-enriched DMEs are robustly correlated between paired tissue and EVs. The robust detection of protein markers related to drug PD profile in MAFLD opens the possibility to track within-subject changes in MAFLD and lays the foundation for future development of a liver-derived EV liquid biopsy to assess markers of drug exposure and response in vivo.

Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.

Delineating the shape of COat Protein complex-II coated membrane bud.

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Extracellular vesicles: A potential new way to assess cholestasis.

Extracellular vesicles (EVs) are small, nonreplicating, lipid-encapsulated nanoparticles that carry protein and nucleic acid cargo derived from their tissue of origin. Due to their capacity to provide comparable insights to solid organ biopsy through a minimally invasive collection procedure, EVs provide an attractive biomarker source. This review will provide an insight, how EVs in circulation may provide a novel way to assess cholestasis and will address the possibility of getting a better understanding of the mechanisms of cholestasis of pregnancy through the use of serial hepatic-specific EVs as a window.

Th2 cell extracellular vesicles promote eosinophil survival through the cytokine cargo IL-3 and prolong airway eosinophilia.

Extracellular vesicles (EVs) mediate intercellular communication during immune responses. EVs are abundant in respiratory biofluids, and the composition of EVs in the lung changes during inflammation. We aimed to quantify the contribution of T cells to airway EVs in allergic lung inflammation and ascertain their function during a type 2 inflammatory response. Genetic membrane tagging was combined with single vesicle flow cytometry to quantify T cell EVs in the airways of mice challenged with ovalbumin or house dust mite. EVs were purified from T helper type 2 (Th2) cell cultures and their functions on eosinophils assessed by flow cytometry and RNA sequencing. Th2 cell EVs were instilled into the lungs of mice to determine effects on lung eosinophilia. Finally, the function of an EV protein cargo was tested using inhibitors and blocking antibodies. T cell EVs are increased in the airways of mice with induced allergic inflammation. EVs secreted by Th2 cells inhibit apoptosis and induce activating pathways in eosinophils in vitro. This effect depends on re-stimulation through the T cell receptor. Th2 cell EVs prolong eosinophilia in vivo during allergic airway inflammation. Th2 cell EVs carry a potent form of the cytokine IL-3 on their surfaces, which inhibits apoptosis by activating Jak1/2-dependent pro-survival programs in eosinophils. Th2 cell EVs promote eosinophil survival and prolong eosinophilia during allergic airway inflammation. This function depends on the EV cargo IL-3, supporting a role for EVs as vehicles of cytokine-based communication in lung inflammation. T cells secrete extracellular vesicles in the airway during allergic lung inflammation.Th2 cell extracellular vesicles inhibit eosinophil apoptosis and prolong airway eosinophilia during allergic lung inflammation.IL-3 carried on Th2 cell EVs is a functional cargo, supporting a role for cytokine-carrying EVs as drivers of type 2 inflammation. This study supports that T cell extracellular vesicles may be important drivers of eosinophilic inflammation through the cytokine cargo IL-3, offering new insights into pro-inflammatory signaling in the allergic lung of patients with asthma.

PACS-1 variant protein is aberrantly localized in Caenorhabditis elegans model of PACS1/PACS2 syndromes.

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

Do extracellular vesicles have specific target cells?; Extracellular vesicle mediated embryo maternal communication.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

Extracellular vesicles released from microglia after palmitate exposure impact brain function

Dietary patterns that include an excess of foods rich in saturated fat are associated with brain dysfunction. Although microgliosis has been proposed to play a key role in the development of brain dysfunction in diet-induced obesity (DIO), neuroinflammation with cytokine over-expression is not always observed. Thus, mechanisms by which microglia contribute to brain impairment in DIO are uncertain. Using the BV2 cell model, we investigated the gliosis profile of microglia exposed to palmitate (200 µmol/L), a saturated fatty acid abundant in high-fat diet and in the brain of obese individuals. We observed that microglia respond to a 24-hour palmitate exposure with increased proliferation, and with a metabolic network rearrangement that favors energy production from glycolysis rather than oxidative metabolism, despite stimulated mitochondria biogenesis. In addition, while palmitate did not induce increased cytokine expression, it modified the protein cargo of released extracellular vesicles (EVs). When administered intra-cerebroventricularly to mice, EVs secreted from palmitate-exposed microglia in vitro led to memory impairment, depression-like behavior, and glucose intolerance, when compared to mice receiving EVs from vehicle-treated microglia. We conclude that microglia exposed to palmitate can mediate brain dysfunction through the cargo of shed EVs.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e.,"cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Unraveling Chylomicron Retention Disease Enhances Insight into SAR1B GTPase Functions and Mechanisms of Actions, While Shedding Light of Intracellular Chylomicron Trafficking.

Over the past three decades, significant efforts have been focused on unraveling congenital intestinal disorders that disrupt the absorption of dietary lipids and fat-soluble vitamins. The primary goal has been to gain deeper insights into intra-enterocyte sites, molecular steps, and crucial proteins/regulatory pathways involved, while simultaneously identifying novel therapeutic targets and diagnostic tools. This research not only delves into specific and rare malabsorptive conditions, such as chylomicron retention disease (CRD), but also contributes to our understanding of normal physiology through the utilization of cutting-edge cellular and animal models alongside advanced research methodologies. This review elucidates how modern techniques have facilitated the decoding of CRD gene defects, the identification of dysfunctional cellular processes, disease regulatory mechanisms, and the essential role of coat protein complex II-coated vesicles and cargo receptors in chylomicron trafficking and endoplasmic reticulum (ER) exit sites. Moreover, experimental approaches have shed light on the multifaceted functions of SAR1B GTPase, wherein loss-of-function mutations not only predispose individuals to CRD but also exacerbate oxidative stress, inflammation, and ER stress, potentially contributing to clinical complications associated with CRD. In addition to dissecting the primary disease pathology, genetically modified animal models have emerged as invaluable assets in exploring various ancillary aspects, including responses to environmental challenges such as dietary alterations, gender-specific disparities in disease onset and progression, and embryonic lethality or developmental abnormalities. In summary, this comprehensive review provides an in-depth and contemporary analysis of CRD, offering a meticulous examination of the CRD current landscape by synthesizing the latest research findings and advancements in the field.

VARP binds SNX27 to promote endosomal supercomplex formation on membranes.

Multiple essential membrane trafficking pathways converge at endosomes to maintain cellular homeostasis by sorting critical transmembrane cargo proteins to the plasma membrane or the trans-Golgi network (TGN). The Retromer heterotrimer (VPS26/VPS35/VPS29 subunits) binds multiple sorting nexin (SNX) proteins on endosomal membranes, but molecular mechanisms regarding formation and regulation of metazoan SNX/Retromer complexes have been elusive. Here, we combine biochemical and biophysical approaches with AlphaFold2 Multimer modeling to identify a direct interaction between the VARP N-terminus and SNX27 PDZ domain. VARP and SNX27 interact with high nanomolar affinity using the binding pocket established for PDZ binding motif (PDZbm) cargo. Specific point mutations in VARP abrogate the interaction in vitro. We further establish a full biochemical reconstitution system using purified mammalian proteins to directly and systematically test whether multiple endosomal coat complexes are recruited to membranes to generate tubules. We successfully use purified coat components to demonstrate which combinations of Retromer with SNX27, ESCPE-1 (SNX2/SNX6), or both complexes can remodel membranes containing physiological cargo motifs and phospholipid composition. SNX27, alone and with Retromer, induces tubule formation in the presence of PI(3)P and PDZ cargo motifs. ESCPE-1 deforms membranes enriched with Folch I and CI-MPR cargo motifs, but surprisingly does not recruit Retromer. Finally, we find VARP is required to reconstitute a proposed endosomal "supercomplex" containing SNX27, ESCPE-1, and Retromer on PI(3)P-enriched membranes. These data suggest VARP functions as a key regulator in metazoans to promote cargo sorting out of endosomes.