Search Results (33)

Based on observations that HIV-1 envelope (Env) proteins on the surfaces of cells have the capacity to fuse with neighboring cells or enveloped viruses that express CD4 receptors and CXCR4 co-receptors, we tested factors that affect the capacities of lentiviral vectors pseudotyped with CD4 and CXCR4 variants to infect Env-expressing cells. The process, which we refer to as fusion in reverse, involves the binding and activation of cellular Env proteins to fuse membranes with lentiviruses carrying CD4 and CXCR4 proteins. We have found that infection via fusion in reverse depends on cell surface Env levels, is inhibitable by an HIV-1-specific fusion inhibitor, and preferentially requires lentiviral pseudotyping with a glycosylphosphatidylinositol (GPI)-anchored CD4 variant and a cytoplasmic tail-truncated CXCR4 protein. We have demonstrated that latently HIV-1-infected cells can be specifically infected using this mechanism, and that activation of latently infected cells increases infection efficiency. The fusion in reverse approach allowed us to characterize how alteration of CD4 plus CXCR4 lipid membranes affected Env protein activities. In particular, we found that perturbation of membrane cholesterol levels did not affect Env activity. In contrast, viruses assembled in cells deficient for long-chain sphingolipids showed increased infectivities, while viruses that incorporated a lipid scramblase were non-infectious. Our results yield new insights into factors that influence envelope protein functions. Full article

GET3 is an ATPase protein that plays a pivotal role in the guided entry of the tail-anchored (GET) pathway. The protein facilitates the targeting and inserting of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) by interacting with a receptor protein complex on the ER. The role of GET3 in various biological processes has been established in yeast, plants, and mammals but not in filamentous fungi. Fusarium graminearum is the major causal agent of Fusarium head blight (FHB), posing a threat to the yield and quality of wheat. In this study, we found that FgGET3 exhibits a high degree of sequence and structural conservation with its homologs across a wide range of organisms. Ectopic expression of FgGET3 in yeast restored the growth defects of the Saccharomyces cerevisiae ScGET3 knock-out mutant. Furthermore, FgGET3 was found to dimerize and localize to the cytoplasm, similar to its homologs in other species. Deletion of FgGET3 in F. graminearum results in decreased fungal growth, fragmented vacuoles, altered abiotic stress responses, reduced conidia production, delayed conidial germination, weakened virulence on wheat spikes and reduced DON production. Collectively, these findings underscore the critical role of FgGET3 in regulating diverse cellular and biological functions essential for the growth and virulence of F. graminearum. Full article

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming. Full article

The Hsp70 family of molecular chaperones acts as a central ‘hub’ in the cell that interacts with numerous newly synthesized proteins to assist in their biogenesis. Apart from its central and well-established role in facilitating protein folding, Hsp70s also act as key decision points in the cellular chaperone network that direct client proteins to distinct biogenesis and quality control pathways. In this paper, we review accumulating data that illustrate a new branch in the Hsp70 network: the post-translational targeting of nascent membrane and organellar proteins to diverse cellular organelles. Work in multiple pathways suggests that Hsp70, via its ability to interact with components of protein targeting and translocation machineries, can initiate elaborate substrate relays in a sophisticated cascade of chaperones, cochaperones, and receptor proteins, and thus provide a mechanism to safeguard and deliver nascent membrane proteins to the correct cellular membrane. We discuss the mechanistic principles gleaned from better-studied Hsp70-dependent targeting pathways and outline the observations and outstanding questions in less well-studied systems. Full article

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe₂₄₁ residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries. Full article

Bacteriophages, or phages, are the most abundant biological entities on Earth. They possess molecular nanodevices to package and store their genome, as well as to introduce it into the cytoplasm of their bacterial prey. Successful phage infection commences with specific recognition of, and adhesion to, a suitable host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limit their structural analyses by experimental approaches. The protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of adhesion devices from siphophages belonging to the P335 group infecting Lactococcus spp., one of the most extensively applied lactic acid bacteria in dairy fermentations. The predictions of representative adhesion devices from types I-IV P335 phages illustrate their very diverse topology. Adhesion devices from types III and IV phages share a common topology with that of Skunavirus p2, with a receptor binding protein anchored to the virion by a distal tail protein loop. This suggests that they exhibit an activation mechanism similar to that of phage p2 prior to host binding. Full article

Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals. Full article

Proteins can be targeted to organellar membranes by using a tail anchor (TA), a stretch of hydrophobic amino acids found at the polypeptide carboxyl-terminus. The Fis1 protein (Fis1p), which promotes mitochondrial and peroxisomal division in the yeast Saccharomyces cerevisiae, is targeted to those organelles by its TA. Substantial evidence suggests that Fis1p insertion into the mitochondrial outer membrane can occur without the need for a translocation machinery. However, recent findings raise the possibility that Fis1p insertion into mitochondria might be promoted by a proteinaceous complex. Here, we have performed atomistic and coarse-grained molecular dynamics simulations to analyze the adsorption, conformation, and orientation of the Fis1(TA). Our results support stable insertion at the mitochondrial outer membrane in a monotopic, rather than a bitopic (transmembrane), configuration. Once inserted in the monotopic orientation, unassisted transition to the bitopic orientation is expected to be blocked by the highly charged nature of the TA carboxyl-terminus and by the Fis1p cytosolic domain. Our results are consistent with a model in which Fis1p does not require a translocation machinery for insertion at mitochondria. Full article

This study aimed to comparatively elucidate the composition structure and techno-functionality of flaxseed protein isolate (FPI), globulin (FG), and albumin (FA) fractions. The results showed that FA possessed smaller particle dimensions and superior protein solubility compared to that of FG (p < 0.05) due to the lower molecular weight and hydrophobicity. FA and FG manifested lamellar structure and nearly spherical morphology, respectively, whereas FPI exhibited small lamellar strip structure packed by the blurring spheres. The Far-UV CD, FTIR spectrum, and intrinsic fluorescence confirmed more flexible conformation of FA than that of FG, followed by FPI. The preferential retention of free phenolic acids was observed for FA, leading to excellent antioxidant activities compared with that of FG in FPI (p < 0.05). FA contributed to the foaming properties of FPI, relying on the earlier interfacial adsorption and higher viscoelastic properties. FA displayed favorable emulsifying capacity but inferior stability due to the limited interfacial adsorption and deformation, as well as loose/porous interface. By comparison, an interlayer anchoring but no direct interface coating was observed for lipid droplets constructed by FG, thereby leading to preferable emulsion stability. However, FPI produced lipid droplets with dense interface owing to the effective migration of FA and FG from bulk phase, concomitant with the easy flocculation and coalescence. Thus, the techno-functionality of flaxseed protein could be tailed by modulating the retention of albumin fraction and specific phenolic acids. Full article

Non-parasitic flatworms are known to temporarily attach to the substrate by secreting a multicomponent bioadhesive to counteract water movements. However, to date, only species of two higher-level flatworm taxa (Macrostomorpha and Proseriata) have been investigated for their adhesive proteins. Remarkably, the surface-binding protein is not conserved between flatworm taxa. In this study, we sequenced and assembled a draft genome, as well as a transcriptome, and generated a tail-specific positional RNA sequencing dataset of the polyclad Theama mediterranea. This led to the identification of 15 candidate genes potentially involved in temporary adhesion. Using in situ hybridisation and RNA interference, we determined their expression and function. Of these 15 genes, 4 are homologues of adhesion-related genes found in other flatworms. With this work, we provide two novel key components on the flatworm temporary adhesion system. First, we identified a Kringle-domain-containing protein (Tmed-krg1), which was expressed exclusively in the anchor cell. This in silico predicted membrane-bound Tmed-krg1 could potentially bind to the cohesive protein, and a knockdown led to a non-adhesive phenotype. Secondly, a secreted tyrosinase (Tmed-tyr1) was identified, which might crosslink the adhesive proteins. Overall, our findings will contribute to the future development of reversible synthetic glues with desirable properties for medical and industrial applications. Full article

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections. Full article

Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138. Full article

Cellular prion protein (PrP^C) is a glycosylphosphatidylinositol (GPI)-anchored protein most abundantly found in the outer membrane of neurons. Due to structural characteristics (a flexible tail and structured core), PrP^C interacts with a wide range of partners. Although PrP^C has been proposed to be involved in many physiological functions, only peripheral nerve myelination homeostasis has been confirmed as a bona fide function thus far. PrP^C misfolding causes prion diseases and PrP^C has been shown to mediate β-rich oligomer-induced neurotoxicity in Alzheimer’s and Parkinson’s disease as well as neuroprotection in ischemia. Upon proteolytic cleavage, PrP^C is transformed into released and attached forms of PrP that can, depending on the contained structural characteristics of PrP^C, display protective or toxic properties. In this review, we will outline prion protein and prion protein fragment properties as well as overview their involvement with interacting partners and signal pathways in myelination, neuroprotection and neurodegenerative diseases. Full article

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease. Full article

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments. Full article