Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Host heparan sulfate promotes ACE2 super-cluster assembly and enhances SARS-CoV-2-associated syncytium formation.

SARS-CoV-2 infection causes spike-dependent fusion of infected cells with ACE2 positive neighboring cells, generating multi-nuclear syncytia that are often associated with severe COVID. To better elucidate the mechanism of spike-induced syncytium formation, we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical stimulator for spike-induced cell-cell fusion. We show that HS binds spike and promotes spike-induced ACE2 clustering, forming synapse-like cell-cell contacts that facilitate fusion pore formation between ACE2-expresing and spike-transfected human cells. Chemical or genetic inhibition of HS mitigates ACE2 clustering, and thus, syncytium formation, whereas in a cell-free system comprising purified HS and lipid-anchored ACE2, HS stimulates ACE2 clustering directly in the presence of spike. Furthermore, HS-stimulated syncytium formation and receptor clustering require a conserved ACE2 linker distal from the spike-binding site. Importantly, the cell fusion-boosting function of HS can be targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice. Thus, HS, as a host factor exploited by SARS-CoV-2 to facilitate receptor clustering and a stimulator of infection-associated syncytium formation, may be a promising therapeutic target for severe COVID.

Heparan sulfate promotes ACE2 super-cluster assembly to enhance SARS-CoV-2-associated syncytium formation.

The mechanism of syncytium formation, caused by spike-induced cell-cell fusion in severe COVID-19, is largely unclear. Here we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical host factor exploited by SARS-CoV-2 to enhance spike’s fusogenic activity. HS binds spike to facilitate ACE2 clustering, generating synapse-like cell-cell contacts to promote fusion pore formation. ACE2 clustering, and thus, syncytium formation is significantly mitigated by chemical or genetic elimination of cell surface HS, while in a cell-free system consisting of purified HS, spike, and lipid-anchored ACE2, HS directly induces ACE2 clustering. Importantly, the interaction of HS with spike allosterically enables a conserved ACE2 linker in receptor clustering, which concentrates spike at the fusion site to overcome fusion-associated activity loss. This fusion-boosting mechanism can be effectively targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice.

Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPα mutants causes lipofuscin accumulation.

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

UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis.

Protein biogenesis at the endoplasmic reticulum (ER) in eukaryotic cells is monitored by a protein quality control system named ER-associated protein degradation (ERAD). While there has been substantial progress in understanding how ERAD eliminates defective polypeptides generated from erroneous folding, how cells remove nascent chains stalled in the translocon during co-translational protein insertion into the ER is unclear. Here we show that ribosome stalling during protein translocation induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24) at the ER. Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation into the ER is safeguarded by a UFMylation-dependent protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans.

Secretion of misfolded cytosolic proteins from mammalian cells is independent of chaperone-mediated autophagy.

In eukaryotic cells, elimination of misfolded proteins is essential for maintaining protein homeostasis and cell viability. Misfolding-associated protein secretion (MAPS) is a protein quality-control mechanism that exports misfolded cytosolic proteins via a compartment characteristic of late endosomes, but how cytosolic proteins enter this compartment is unclear. Because chaperone-mediated autophagy (CMA) is a known mechanism that imports cytosolic proteins bearing a specific CMA motif to lysosomes for degradation and because late endosomes and lysosomes overlap significantly in mammalian cells, we determined here whether CMA is involved in targeting protein cargoes to the lumen of late endosomes in MAPS. Using HEK293T and COS-7 cells and immunoblotting and -staining and coimmunoprecipitation methods, we show that, unlike CMA, the secretion of misfolded proteins in MAPS does not require cargo unfolding, is inhibited by serum starvation, and is not dependent on the CMA motif in cargo. Intriguingly, knockdown of lysosome-associated membrane protein 2 (LAMP2), which consists of three isoforms, including a variant proposed to form a protein channel on lysosomes for CMA, attenuated MAPS. However, this could not be attributed to the proposed channel function of the LAMP2a isoform because overexpression of a cytosolic MAPS stimulator, DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5), fully rescued the secretion defect associated with LAMP2 deficiency. We conclude that, in MAPS, cargoes use a CMA-independent mechanism to enter a nondegradative prelysosomal compartment.

DNAJC5 facilitates USP19-dependent unconventional secretion of misfolded cytosolic proteins.

Cell-to-cell transmission of misfolded proteins propagates proteotoxic stress in multicellular organisms when transmitted polypeptides serve as a seeding template to cause protein misfolding in recipient cells, but how misfolded proteins are released from cells to initiate this process is unclear. Misfolding-associated protein secretion (MAPS) is an unconventional protein-disposing mechanism that specifically exports misfolded cytosolic proteins including various neurodegenerative disease-causing proteins. Here we establish the HSC70 co-chaperone DNAJC5 as an essential mediator of MAPS. USP19, a previously uncovered MAPS regulator binds HSC70 and acts upstream of HSC70 and DNAJC5. We further show that as a membrane-associated protein localized preferentially to late endosomes and lysosomes, DNAJC5 can chaperone MAPS client proteins to the cell exterior. Intriguingly, upon secretion, misfolded proteins can be taken up through endocytosis and eventually degraded in the lysosome. Collectively, these findings suggest a transcellular protein quality control regulatory pathway in which a deubiquitinase-chaperone axis forms a "triaging hub", transferring aberrant polypeptides from stressed cells to healthy ones for disposal.

Crystal structure and allosteric activation of protein kinase C ßII.

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.

Hybrid structural model of the complete human ESCRT-0 complex.

The human Hrs and STAM proteins comprise the ESCRT-0 complex, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. Here we report a model for the complete ESCRT-0 complex based on the crystal structure of the Hrs-STAM core complex, previously solved domain structures, hydrodynamic measurements, and Monte Carlo simulations. ESCRT-0 expressed in insect cells has a hydrodynamic radius of RH = 7.9 nm and is a 1:1 heterodimer. The 2.3 Angstroms crystal structure of the ESCRT-0 core complex reveals two domain-swapped GAT domains and an antiparallel two-stranded coiled-coil, similar to yeast ESCRT-0. ESCRT-0 typifies a class of biomolecular assemblies that combine structured and unstructured elements, and have dynamic and open conformations to ensure versatility in target recognition. Coarse-grained Monte Carlo simulations constrained by experimental RH values for ESCRT-0 reveal a dynamic ensemble of conformations well suited for diverse functions.

Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin.

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.

[3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP.

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP. PDE5 was the only [3H]sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [3H]sildenafil exchange dissociation yielded two components with t1/2 values of 1 and 14 min and corresponding calculated KD values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [3H]Sildenafil binding isotherm of PDE5 indicated KD was 8.3 to 13.3 nM, and low cGMP decreased the KD to 4.8 nM but only slightly increased Bmax to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.

Structure of the GAT domain of human GGA1: a syntaxin amino-terminal domain fold in an endosomal trafficking adaptor.

The Golgi-associated, gamma-adaptin homologous, ADP-ribosylation factor (ARF)-interacting proteins (GGAs) are adaptors that sort receptors from the trans-Golgi network into the endosomallysosomal pathway. The GGAs and TOM1 (GAT) domains of the GGAs are responsible for their ARF-dependent localization. The 2.4-A crystal structure of the GAT domain of human GGA1 reveals a three-helix bundle, with a long N-terminal helical extension that is not conserved in GAT domains that do not bind ARF. The ARF binding site is located in the N-terminal extension and is separate from the core three-helix bundle. An unanticipated structural similarity to the N-terminal domain of syntaxin 1a was discovered, comprising the entire three-helix bundle. A conserved binding site on helices 2 and 3 of the GAT domain three-helix bundle is predicted to interact with coiled-coil-containing proteins. We propose that the GAT domain is descended from the same ancestor as the syntaxin 1a N-terminal domain, and that both protein families share a common function in binding coiled-coil domain proteins.

Structural genomics and signaling domains.

Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.