Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

Programmable Extracellular Vesicles for Macromolecule Delivery and Genome Modifications.

Getting large macromolecules through the plasma membrane and endosomal barriers remains a major challenge. Here, we report a generalizable method of delivering proteins and ribonucleoproteins (RNPs) to cells in vitro and mouse liver tissue in vivo with engineered ectosomes. These ectosomes, referred to as "Gectosomes," are designed to co-encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. We found that this method enables active cargo loading, improves the specific activity of cargo delivery, and facilitates Gectosome purification. Experimental and mathematical modeling analyses suggest that active cargo loading reduces non-specific encapsulation of cellular proteins, particularly nucleic-acid-binding proteins. Using Gectosomes that encapsulate Cre, Ago2, and SaCas9, we demonstrate their ability to execute designed modifications of endogenous genes in cell lines in vitro and mouse liver tissue in vivo, paving the way toward applications of this technology for the treatment of a wide range of human diseases.

PBRM1 and the glycosylphosphatidylinositol biosynthetic pathway promote tumor killing mediated by MHC-unrestricted cytotoxic lymphocytes.

Major histocompatibility complex (MHC)-unrestricted cytotoxic lymphocytes (CLs) such as natural killer (NK) cells can detect and destroy tumor and virus-infected cells resistant to T cell-mediated killing. Here, we performed genome-wide genetic screens to identify tumor-intrinsic genes regulating killing by MHC-unrestricted CLs. A group of genes identified in our screens encode enzymes for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is not involved in tumor response to T cell-mediated cytotoxicity. Another gene identified in the screens was PBRM1, which encodes a subunit of the PBAF form of the SWI/SNF chromatin-remodeling complex. PBRM1 mutations in tumor cells cause resistance to MHC-unrestricted killing, in contrast to their sensitizing effects on T cell-mediated killing. PBRM1 and the GPI biosynthetic pathway regulate the ligands of NK cell receptors in tumor cells and promote cytolytic granule secretion in CLs. The regulators identified in this work represent potential targets for cancer immunotherapy.

Filterless radio-over-fiber system based on polarization multiplexing to generate an 80 GHz millimeter wave.

We propose a filterless full-duplex radio-over-fiber system based on polarization multiplexing and demonstrate the generation of an 80 GHz millimeter wave using two Mach-Zehnder modulators. By adjusting the polarization direction, we could generate an 80 GHz frequency millimeter-wave signal and restore the original pure light carrier, providing a light source for the uplink. The simulation results show that the 80 GHz millimeter-wave signal was obtained with a 23.48 dB radio-frequency sideband suppression ratio. Furthermore, we showed that the proposed scheme is relatively flexible and free from the limitation of filter fixed bandwidth in addition to being simple and economical.

Delivery of a model lipophilic membrane cargo to bone marrow via cell-derived microparticles.

Bone marrow (BM) is the central immunological organ and the origin of hematological diseases. Efficient and specific drug delivery to the BM is an unmet need. We tested delivery of fluorescent indocarbocyanine lipids (ICLs, DiR, DiD, DiI) as a model lipophilic cargo, via different carriers. Systemically injected T-lymphocyte cell line Jurkat delivered ICLs to the BM more efficiently than erythrocytes, and more selectively than PEGylated liposomes. Near infrared imaging showed that the delivery was restricted to the BM, lungs, liver and spleen, with no accumulation in the kidneys, brain, heart, intestines, fat tissue and pancreas. Following systemic injection of ICL-labeled cells in immunodeficient or immunocompetent mice, few cells arrived in the BM intact. However, between 5 and 10% of BM cells were ICL-positive. Confocal microscopy of intact BM confirmed that ICLs are delivered independently of the injected cells. Flow cytometry analysis showed that the lipid accumulated in both CD11b + and CD11b- cells, and in hematopoietic progenitors. In a xenograft model of acute myeloid leukemia, a single injection of 10 million Jurkat cells delivered DiD to ~15% of the tumor cells. ICL-labeled cells disappeared from blood almost immediately post-intravenous injection, but numerous cell-derived microparticles continued to circulate in blood. The microparticle particle formation was not due to the ICL labeling or complement attack and was observed after injection of both syngeneic and xenogeneic cells. Injection of microparticles produced in vitro from Jurkat cells resulted in a similar ICL delivery as the injection of intact Jurkat cells. Our results demonstrate a novel delivery paradigm wherein systemically injected cells release microparticles that accumulate in the BM. In addition, the results have important implications for studies involving systemically administered cell therapies.

A myosin-7B-dependent endocytosis pathway mediates cellular entry of α-synuclein fibrils and polycation-bearing cargos.

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE.

Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.

AAGAB Controls AP2 Adaptor Assembly in Clathrin-Mediated Endocytosis.

Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.

The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis.

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

Self-marked HCN gas based FBG demodulation in thermal cycling process for aerospace environment.

The thermal cycling process experienced by spacecraft during orbital operation would lead to deterioration of the demodulation performance of fiber Bragg grating (FBG). A new demodulation method based on Fabry-Perot (F-P) filter and hydrogen cyanide (HCN) gas is proposed to improve the performance. The method skillfully utilizes the self-marked HCN absorption lines as absolute wavelength references. In the thermal cycling environment whose temperature ranging from 5°Cto 65°C,the fluctuation of demodulation wavelength reduces to ± 2.6 pm, which is improved by 3.1 times compared with traditional method. The proposed method also shows a good robustness in the cases of weak light source intensity and poor signal-to-noise ratio (SNR) of HCN spectrum.

SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.

RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.

Rab GTPases are switched from their GDP-bound inactive conformation to a GTP-bound active state by guanine nucleotide exchange factors (GEFs). The first putative GEFs isolated for Rabs are RABIF (Rab-interacting factor)/MSS4 (mammalian suppressor of Sec4) and its yeast homolog DSS4 (dominant suppressor of Sec4). However, the biological function and molecular mechanism of these molecules remained unclear. In a genome-wide CRISPR genetic screen, we isolated RABIF as a positive regulator of exocytosis. Knockout of RABIF severely impaired insulin-stimulated GLUT4 exocytosis in adipocytes. Unexpectedly, we discovered that RABIF does not function as a GEF, as previously assumed. Instead, RABIF promotes the stability of Rab10, a key Rab in GLUT4 exocytosis. In the absence of RABIF, Rab10 can be efficiently synthesized but is rapidly degraded by the proteasome, leading to exocytosis defects. Strikingly, restoration of Rab10 expression rescues exocytosis defects, bypassing the requirement for RABIF. These findings reveal a crucial role of RABIF in vesicle transport and establish RABIF as a Rab-stabilizing holdase chaperone, a previously unrecognized mode of Rab regulation independent of its GDP-releasing activity. Besides Rab10, RABIF also regulates the stability of two other Rab GTPases, Rab8 and Rab13, suggesting that the requirement of holdase chaperones is likely a general feature of Rab GTPases.

Comparative Haploid Genetic Screens Reveal Divergent Pathways in the Biogenesis and Trafficking of Glycophosphatidylinositol-Anchored Proteins.

Glycophosphatidylinositol-anchored proteins (GPI-APs) play essential roles in physiology, but their biogenesis and trafficking have not been systematically characterized. Here, we took advantage of the recently available haploid genetics approach to dissect GPI-AP pathways in human cells using prion protein (PrP) and CD59 as model molecules. Our screens recovered a large number of common and unexpectedly specialized factors in the GPI-AP pathways. PIGN, PGAP2, and PIGF, which encode GPI anchor-modifying enzymes, were selectively isolated in the CD59 screen, suggesting that GPI anchor composition significantly influences the biogenesis of GPI-APs in a substrate-dependent manner. SEC62 and SEC63, which encode components of the ER-targeting machinery, were selectively recovered in the PrP screen, indicating that they do not constitute a universal route for the biogenesis of mammalian GPI-APs. Together, these comparative haploid genetic screens demonstrate that, despite their similarity in overall architecture and subcellular localization, GPI-APs follow markedly distinct biosynthetic and trafficking pathways.

The N- and C-terminal domains of tomosyn play distinct roles in soluble N-ethylmaleimide-sensitive factor attachment protein receptor binding and fusion regulation.

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion.

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H(abc) domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H(abc) domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H(abc) domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q(bc) configuration. We found that neither the linker nor the Q(bc) configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.

Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response.

Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.