Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Novel oxindole compounds inhibit the aggregation of amyloidogenic proteins associated with neurodegenerative diseases

Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on amyloidogenic protein aggregation. However, no effective drug suitable for transition to clinical use has been found. Here we examined several novel oxindole compounds (GIF compounds) for their inhibitory effects on aggregate formation of the canine mutant superoxide dismutase 1 (cSOD1 E40K), a causative mutation resulting in DM, using Thioflavin-T fluorescence. Most GIF compounds inhibited the aggregation of cSOD1 E40K. Among the compounds, GIF-0854-r and GIF-0890-r were most effective. Their inhibitory effects were also observed in cSOD1 E40K-transfected cells. Additionally, GIF-0890-r effectively inhibited the aggregate formation of human SOD1 G93A, a causative mutation of ALS. GIF-0827-r and GIF-0856-r also effectively inhibited aggregate formation of human prion protein (hPrP). Subsequently, the correlation between their inhibitory effects on cSOD1 and hPrP aggregation was shown, indicating GIF compounds inhibited the aggregate formation of multiple amyloidogenic proteins. Conclusively, the novel oxindole compounds (GIF-0827-r, GIF-0854-r, GIF-0856-r, and GIF-0890-r) are proposed as useful therapeutic candidates for amyloidogenic neurodegenerative disorders.     

A Conservative Point Mutation in a Dynamic Antigen-binding Loop of Human Immunoglobulin λ6 Light Chain Promotes Pathologic Amyloid Formation

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ∼30 Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.     

Revisiting misfolding propensity of serum amyloid A1: Special focus on the signal peptide region

AA amyloidosis is the result of overproduction and aberrant processing of acute-phase serum amyloid A1 (SAA1) by hepatocytes. Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein undergoes a cleavage of 18 residues consisting of the signal peptide at the N-terminal region. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region. We first examined the signal peptide amino acid SAA derived from different animal species. A library of 16 peptides was designed to evaluate the propensity of aggregation. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using in silico Tango program, thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and seeding with misfolded human SAA1 signal peptide. After 7 days of incubation, most of the SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 100 μM in 50 mM Tris buffer at 37 °C by TEM. All peptides were able to generate fibrils at a higher concentration, i.e 500 μM in 25 mM Tris buffer with 50% HFIP, by ThT. All SAA1 signal synthetic peptides designed from the different animal species had the propensity to misfold and form fibrils, particularly in species with low occurrence of systemic amyloidosis. The human SAA1 signal peptide region was capable to seed the SAA1 1–25 and 32–47 peptide regions. Characterizing fibrillar conformations are relevant for seeding intact and/or fragmented SAA, which may contribute, to the mechanism of protein misfolding. This research signifies the importance of the signal peptide region and its possible contribution to the misfolding of aggregation-prone proteins.     

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.     

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.     

Nucleic acid actions on abnormal protein aggregation,phase transitions and phase separation

Liquid–liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.     

Structural Insights into the Mechanism of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag–PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P₂ than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(–)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(–)MA binds to PI(4,5)P₂ via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA–IP binding is governed by charge–charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P₂ and phosphatidylserine enhance myr(–)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(–)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly.     

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.     

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.     

Neuronal growth regulator 1 promotes adipocyte lipid trafficking via interaction with CD36

Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in situ proximal ligation assays indicated that NEGR1-CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the white adipose tissue and liver tissues of Negr1^?/? mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1^?/? mouse embryonic fibroblasts showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control mouse embryonic fibroblasts. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.     

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.     

Fucoidan exerts antidepressant-like effects in mice via regulating the stability of surface AMPARs

The inflammatory hypothesis is one of the most important mechanisms of depression. Fucoidan is a bioactive sulfated polysaccharide abundant in brown seaweeds with anti-inflammatory activity. However, the antidepressant effects of fucoidan on chronic stress-induced depressive-like behaviors have not been well elucidated. Here, we used two different depressive-like mouse models, lipopolysaccharide (LPS) and chronic restraint stress (CRS) models, to explore the detailed molecular mechanism underlying its antidepressant-like effects in C57BL/6J mice by combining multiple behavioral, molecular and immunofluorescence experiments. Adenovirus-mediated overexpression of caspase-1 and pharmacological inhibitors were also used to clarify the antidepressant mechanisms of fucoidan. We found that acute administration of fucoidan did not produce antidepressant effects in the tail suspension test (TST) and forced swim test (FST). Interestingly, chronic fucoidan administration not only dose-dependently reduced stress-induced depressive-like behaviors in the TST, FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT), but also alleviated the downregulation of brain-derived neurotrophic factor (BDNF)-dependent synaptic plasticity via inhibiting caspase-1-mediated inflammation in the hippocampus of mice. Moreover, fucoidan significantly ameliorated behavioral and synaptic plasticity abnormalities in the overexpression of caspase-1 in the hippocampus of mice. Furthermore, blocking BDNF abolished the antidepressant-like effects of fucoidan in mice. Therefore, our findings clearly indicate that fucoidan provides a potential supplementary noninvasive treatment for depression by inhibition of hippocampal inflammation.