Human Peroxin PEX3 Is Co-translationally Integrated into the ER and Exits the ER in Budding Vesicles.

The long-standing paradigm that all peroxisomal proteins are imported post-translationally into pre-existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co-translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N-terminal transmembrane segment (TMS) of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3-containing ribosome•nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP-dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.

Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility.

The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.

The C-terminal Domains of Apoptotic BH3-only Proteins Mediate Their Insertion into Distinct Biological Membranes.

Changes in the equilibrium of pro- and anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family in the mitochondrial outer membrane (MOM) induce structural changes that commit cells to apoptosis. Bcl-2 homology-3 (BH3)-only proteins participate in this process by either activating pro-apoptotic effectors or inhibiting anti-apoptotic components and by promoting MOM permeabilization. The association of BH3-only proteins with MOMs is necessary for the activation and amplification of death signals; however, the nature of this association remains controversial, as these proteins lack a canonical transmembrane sequence. Here we used an in vitro expression system to study the insertion capacity of hydrophobic C-terminal regions of the BH3-only proteins Bik, Bim, Noxa, Bmf, and Puma into microsomal membranes. An Escherichia coli complementation assay was used to validate the results in a cellular context, and peptide insertions were modeled using molecular dynamics simulations. We also found that some of the C-terminal domains were sufficient to direct green fluorescent protein fusion proteins to specific membranes in human cells, but the domains did not activate apoptosis. Thus, the hydrophobic regions in the C termini of BH3-only members associated in distinct ways with various biological membranes, suggesting that a detailed investigation of the entire process of apoptosis should include studying the membranes as a setting for protein-protein and protein-membrane interactions.

Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi.

BACKGROUND: The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3ß1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made. RESULTS: Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions. CONCLUSIONS: Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.

Molecular and topological membrane folding determinants of transient receptor potential vanilloid 2 channel.

Transient Receptor Potential (TRP) channels are related to adaptation to the environment and somatosensation. The transient receptor potential vanilloid (TRPV) subfamily includes six closely evolutionary related ion channels sharing the same domain organization and tetrameric arrangement in the membrane. In this study we have characterized biochemically TRPV2 channel membrane protein folding and transmembrane (TM) architecture. Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) show a key role for this region in targeting the protein to the membrane. We have demonstrated the co-translational insertion of the membrane-embedded region of the TRPV2 and its disposition in biological membranes, identifying that TM1-TM4 and TM5-TM6 regions can assemble as independent folding domains. The ARD is not required for TM domain insertion in the membrane. The folding features observed for TRPV2 may be conserved and shared among other TRP channels outside the TRPV subfamily.

Cetylpyridinium chloride and chlorhexidine show antiviral activity against Influenza A virus and Respiratory Syncytial virus in vitro.

BACKGROUND: The oral cavity is the site of entry and replication for many respiratory viruses. Furthermore, it is the source of droplets and aerosols that facilitate viral transmission. It is thought that appropriate oral hygiene that alters viral infectivity might reduce the spread of respiratory viruses and contribute to infection control. MATERIALS AND METHODS: Here, we analyzed the antiviral activity of cetylpyridinium chloride (CPC), chlorhexidine (CHX), and three commercial CPC and CHX-containing mouthwash preparations against the Influenza A virus and the Respiratory syncytial virus. To do so the aforementioned compounds and preparations were incubated with the Influenza A virus or with the Respiratory syncytial virus. Next, we analyzed the viability of the treated viral particles. RESULTS: Our results indicate that CPC and CHX decrease the infectivity of both the Influenza A virus and the Respiratory Syncytial virus in vitro between 90 and 99.9% depending on the concentration. Likewise, CPC and CHX-containing mouthwash preparations were up to 99.99% effective in decreasing the viral viability of both the Influenza A virus and the Respiratory syncytial virus in vitro. CONCLUSION: The use of a mouthwash containing CPC or CHX alone or in combination might represent a cost-effective measure to limit infection and spread of enveloped respiratory viruses infecting the oral cavity, aiding in reducing viral transmission. Our findings may stimulate future clinical studies to evaluate the effects of CPC and CHX in reducing viral respiratory transmissions.

Cetylpyridinium Chloride-Containing Mouthwashes Show Virucidal Activity against Herpes Simplex Virus Type 1.

The oral cavity is particularly susceptible to viral infections that are self-recovering in most cases. However, complications may appear in severe cases and/or immunocompromised subjects. Cetylpyridinium chloride (CPC)-containing mouthwashes are able to decrease the infectivity of the SARS-CoV-2 virus by disrupting the integrity of the viral envelope. Here, we show that CPC, as the active ingredient contained in commercialized, exerts significant antiviral activity against enveloped viruses, such as HSV-1, but not against non-enveloped viruses, such as HPV. CPC-containing mouthwashes have been used as antiseptics for decades, and thus, they can represent a cost-effective measure to limit infection and spread of enveloped viruses infecting the oral cavity, aiding in reducing viral transmission.

Membrane integration of poliovirus 2B viroporin.

Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.

Cetylpyridinium chloride promotes disaggregation of SARS-CoV-2 virus-like particles.

BACKGROUND: SARS-CoV-2 is continuously disseminating worldwide. The development of strategies to break transmission is mandatory. AIM OF THE STUDY: To investigate the potential of cetylpyridinium chloride (CPC) as a viral inhibitor. METHODS: SARS-CoV-2 Virus Like-Particles (VLPs) were incubated with CPC, a potent surfactant routinely included in mouthwash preparations. RESULTS: Concentrations of 0.05% CPC (w/v) commonly used in mouthwash preparations are sufficient to promote the rupture of SARS-CoV-2 VLP membranes. CONCLUSION: Including CPC in mouthwashes could be a prophylactic strategy to keep SARS-CoV-2 from spreading.

Insertion of Bacteriorhodopsin Helix C Variants into Biological Membranes.

A peptide corresponding to bacteriorhodopsin (bR) helix C, later named pHLIP, inserts across lipid bilayers as a monomeric α-helix at acidic pH, but is an unstructured surface-bound monomer at neutral pH. As a result of such pH-responsiveness, pHLIP targets acidic tumors and has been used as a vehicle for imaging and drug-delivery cargoes. To gain insights about the insertion of bR helix C into biological membranes, we replaced two key aspartic residues that control the topological transition from the aqueous phase into a lipid bilayer. Here, we used an in vitro transcription-translation system to study the translocon-mediated insertion of helix C-derived segments into rough microsomes. Our data provide the first quantitative biological understanding of this effect. Interestingly, replacing the aspartic residues by glutamic residues does not significantly alters the insertion propensity, while replacement by alanines promotes a transmembrane orientation. These results are consistent with mutational data obtained in synthetic liposomes by manipulating pH conditions. Our findings support the notion that the translocon facilitates topogenesis under physiological pH conditions.

Prion soft amyloid core driven self-assembly of globular proteins into bioactive nanofibrils.

Amyloids have been exploited to build amazing bioactive materials. In most cases, short synthetic peptides constitute the functional components of such materials. The controlled assembly of globular proteins into active amyloid nanofibrils is still challenging, because the formation of amyloids implies a conformational conversion towards a ß-sheet-rich structure, with a concomitant loss of the native fold and the inactivation of the protein. There is, however, a remarkable exception to this rule: yeast prions. They are singular proteins able to switch between a soluble and an amyloid state. In both states, the structure of their globular domains remains essentially intact. The transit between these two conformations is encoded in prion domains (PrDs): long and disordered sequences to which the active globular domains are appended. PrDs are much larger than typical self-assembling peptides. This seriously limits their use for nanotechnological applications. We have recently shown that these domains contain soft amyloid cores (SACs) that suffice to nucleate their self-assembly reaction. Here we genetically fused a model SAC with different globular proteins. We demonstrate that this very short sequence acts as a minimalist PrD, driving the selective and slow assembly of the initially soluble fusion proteins into amyloid fibrils in which the globular proteins retain their native structure and display high activity. Overall, we provide here a novel, modular and straightforward strategy to build active protein-based nanomaterials at a preparative scale.

A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation.

Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states.

Transmembrane but not soluble helices fold inside the ribosome tunnel.

Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed ß-sheets, forms ß-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.

Charge pair interactions in transmembrane helices and turn propensity of the connecting sequence promote helical hairpin insertion.

α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

Polar/Ionizable residues in transmembrane segments: effects on helix-helix packing.

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.

N-glycosylation efficiency is determined by the distance to the C-terminus and the amino acid preceding an Asn-Ser-Thr sequon.

N-glycosylation is the most common and versatile protein modification. In eukaryotic cells, this modification is catalyzed cotranslationally by the enzyme oligosaccharyltransferase, which targets the ß-amide of the asparagine in an Asn-Xaa-Ser/Thr consensus sequon (where Xaa is any amino acid but proline) in nascent proteins as they enter the endoplasmic reticulum. Because modification of the glycosylation acceptor site on membrane proteins occurs in a compartment-specific manner, the presence of glycosylation is used to indicate membrane protein topology. Moreover, glycosylation sites can be added to gain topological information. In this study, we explored the determinants of N-glycosylation with the in vitro transcription/translation of a truncated model protein in the presence of microsomes and surveyed 25,488 glycoproteins, of which 2,533 glycosylation sites had been experimentally validated. We found that glycosylation efficiency was dependent on both the distance to the C-terminus and the nature of the amino acid that preceded the consensus sequon. These findings establish a broadly applicable method for membrane protein tagging in topological studies.