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JACS Au Sep 2023The use of nanopores for the single-molecule sensing of folded proteins and biomacromolecules has recently gained attention. Here, we introduce a simplified synthetic...
The use of nanopores for the single-molecule sensing of folded proteins and biomacromolecules has recently gained attention. Here, we introduce a simplified synthetic α-helical transmembrane pore, pPorA, as a nanoreactor and sensor that exhibits functional versatility comparable to that of engineered protein and DNA nanopores. The pore, built from the assembly of synthetic 40-amino-acid-long peptides, is designed to contain cysteine residues within the lumen and at the pore terminus for site-specific chemical modification probed using single-channel electrical recordings. The reaction of the pore with differently charged activated thiol reagents was studied, wherein positively charged reagents electrophoretically driven into the pore resulted in pore blocking in discrete steps upon covalent bond formation. The asymmetric blockage patterns resulting from cis and trans-side addition of reagents reveal the pore orientation in the lipid membrane. Furthermore, activated PEG thiols covalently blocked the pores over a longer duration in a charge-independent manner, establishing the large diameter and orientation of the formed pores. While the covalent binding of thiol reagents caused a drop in the pore conductance, cationic cyclic octasaccharides produced time-resolved translocation events, confirming the structural flexibility and tunability of the pores. The ability of the pore to accommodate large analytes and the considerable current amplitude variation following bond formation events are promising for developing platforms to resolve multistep chemical reactions at the single-molecule level for applications in synthetic nanobiotechnology.
PubMed: 37772177
DOI: 10.1021/jacsau.3c00221 -
The Journal of Biological Chemistry Jul 2021The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies... (Review)
Review
The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.
Topics: Active Transport, Cell Nucleus; DNA Viruses; Humans; Immune Evasion; Immunity, Innate; NF-kappa B; Nuclear Pore; Nuclear Pore Complex Proteins; RNA Viruses; Viral Nonstructural Proteins; Virus Diseases; Virus Replication
PubMed: 34097873
DOI: 10.1016/j.jbc.2021.100856 -
Cells Jan 2022In addition to its structural role in enclosing and protecting the genome, the nuclear envelope (NE) forms a highly adaptive communication interface between the... (Review)
Review
In addition to its structural role in enclosing and protecting the genome, the nuclear envelope (NE) forms a highly adaptive communication interface between the cytoplasm and the nuclear interior in eukaryotic cells. The double membrane of the NE is perforated by nuclear pores lined with large multi-protein structures, called nuclear-pore complexes (NPCs), which selectively allow the bi-directional transport of ions and macromolecular cargo. In order to nucleate a pore, the inner and outer nuclear membrane have to fuse at the site of NPC insertion, a process requiring both lipid bilayers to be deformed into highly curved structures. How this curvature is achieved and which factors are involved in inducing and stabilizing membrane curvature at the nuclear pore remain largely unclear. In this review, we will summarize the molecular mechanisms thought to be involved in membrane curvature generation, with a particular emphasis on the role of lipids and lipid metabolism in shaping the nuclear pore membrane.
Topics: Eukaryotic Cells; Lipid Bilayers; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex Proteins
PubMed: 35159279
DOI: 10.3390/cells11030469 -
Cell Mar 2016Nuclear pore complexes (NPCs) perforate the nuclear envelope and serve as the primary transport gates for molecular exchange between nucleus and cytoplasm. Stripping the... (Review)
Review
Nuclear pore complexes (NPCs) perforate the nuclear envelope and serve as the primary transport gates for molecular exchange between nucleus and cytoplasm. Stripping the megadalton complex down to its most essential organizational elements, one can divide the NPC into scaffold components and the disordered elements attached to them that generate a selective barrier between compartments. These structural elements exhibit flexibility, which may hold a clue in understanding NPC assembly and function. Here we review the current status of NPC research with a focus on the functional implications of its structural and compositional heterogeneity.
Topics: Animals; Evolution, Molecular; Humans; Models, Molecular; Nuclear Pore; Nuclear Pore Complex Proteins
PubMed: 26967283
DOI: 10.1016/j.cell.2016.01.034 -
International Journal of Molecular... Feb 2023Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is... (Review)
Review
Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is their ability to form pores that disrupt the membrane permeability barrier and ion homeostasis and generally induce cell death. Some PFPs are part of the genetically encoded machinery of eukaryotic cells that are activated against infection by pathogens or in physiological programs to carry out regulated cell death. PFPs organize into supramolecular transmembrane complexes that perforate membranes through a multistep process involving membrane insertion, protein oligomerization, and finally pore formation. However, the exact mechanism of pore formation varies from PFP to PFP, resulting in different pore structures with different functionalities. Here, we review recent insights into the molecular mechanisms by which PFPs permeabilize membranes and recent methodological advances in their characterization in artificial and cellular membranes. In particular, we focus on single-molecule imaging techniques as powerful tools to unravel the molecular mechanistic details of pore assembly that are often obscured by ensemble measurements, and to determine pore structure and functionality. Uncovering the mechanistic elements of pore formation is critical for understanding the physiological role of PFPs and developing therapeutic approaches.
Topics: Single Molecule Imaging; Cell Membrane; Porins
PubMed: 36901959
DOI: 10.3390/ijms24054528 -
Biochimica Et Biophysica Acta Mar 2016Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as... (Review)
Review
Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Cell Membrane; Clostridium; Humans; Pore Forming Cytotoxic Proteins; Protein Multimerization
PubMed: 26278641
DOI: 10.1016/j.bbamem.2015.08.006 -
The Protein Journal Aug 2019During my postdoc interview in June of 1998, I asked Günter why he was moving more towards the nucleus in his latest studies. He said, "Well Joe, that's where... (Review)
Review
During my postdoc interview in June of 1998, I asked Günter why he was moving more towards the nucleus in his latest studies. He said, "Well Joe, that's where everything starts." By the end of the interview, I accepted the postdoc. He had a way of making everything sound so cool. Günter's progression was natural, since the endoplasmic reticulum and the nucleus are the only organelles that share the same membrane. The nuclear envelope extends into a double membrane system with nuclear pore complexes embedded in the pore membrane openings. Even while writing this review, I remember Günter stressing; it is the nuclear pore complex. Just saying nuclear pore doesn't encompass the full magnitude of its significance. The nuclear pore complex is one of the largest collection of proteins that fit together for an overall function: transport. This review will cover the Blobel lab contributions in the quest for the blueprint of the nuclear pore complex from isolation of the nuclear envelope and nuclear lamin to the ring structures.
Topics: Animals; Biographies as Topic; Cytoskeleton; Famous Persons; Humans; Lamins; Membrane Glycoproteins; Nuclear Lamina; Nuclear Pore; Nuclear Pore Complex Proteins; Rats; Yeasts
PubMed: 31410705
DOI: 10.1007/s10930-019-09858-z -
Toxins Aug 2018Recent technological advances have seen increasing numbers of complex structures from diverse pore-forming toxins (PFT). The ClyA family of α-PFTs comprises a broad... (Review)
Review
Recent technological advances have seen increasing numbers of complex structures from diverse pore-forming toxins (PFT). The ClyA family of α-PFTs comprises a broad variety of assemblies including single-, two- and three-component toxin systems. With crystal structures available for soluble subunits of all major groups in this extended protein family, efforts now focus on obtaining molecular insights into physiological pore formation. This review provides an up-to-date discussion on common and divergent structural and functional traits that distinguish the various ClyA family PFTs. Open questions of this research topic are outlined and discussed.
Topics: Cell Membrane; Pore Forming Cytotoxic Proteins; Protein Conformation
PubMed: 30142951
DOI: 10.3390/toxins10090343 -
Biochimica Et Biophysica Acta Mar 2016Pore forming proteins (PFPs) share the ability of creating pores that allow the passage of ions, proteins or other constituents through a wide variety of target... (Review)
Review
Pore forming proteins (PFPs) share the ability of creating pores that allow the passage of ions, proteins or other constituents through a wide variety of target membranes, ranging from bacteria to humans. They often cause cell death, as pore formation disrupts the membrane permeability barrier required for maintaining cell homeostasis. The organization into supramolecular complexes or oligomers that pierce the membrane is a common feature of PFPs. However, the molecular pathway of self-assembly and pore opening remains unclear. Here, we review the most recent discoveries in the mechanism of membrane oligomerization and pore formation of a subset of PFPs, the α-PFPs, whose pore-forming domains are formed by helical segments. Only now we are starting to grasp the molecular details of their function, mainly thanks to the introduction of single molecule microscopy and nanoscopy techniques. This article is part of a Special Issue entitled: Pore-forming toxins edited by Mauro Dalla Serra and Franco Gambale.
Topics: Animals; Cell Membrane; Humans; Porins; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 26375417
DOI: 10.1016/j.bbamem.2015.09.013 -
Materials (Basel, Switzerland) Mar 2024Ground Granulated Blast-Furnace Slag (GGBS) and silica fume (SF) are frequently utilized in gel materials to produce environmentally sustainable concrete. The blend of...
Ground Granulated Blast-Furnace Slag (GGBS) and silica fume (SF) are frequently utilized in gel materials to produce environmentally sustainable concrete. The blend of the two components contributes to an enhancement in the pore structure, which, in turn, increases the mechanical strength of the material and the compactness of the pore structure and decreases the permeability, thereby improving the durability of the concrete. In this study, the pore structures of GGBS and SF blends are assessed using Nuclear Magnetic Resonance (NMR) and Mercury Intrusion Porosimetry (MIP) tests. These methodologies provide a comprehensive evaluation of the effect of GGBS and SF on the pore structure of cementitious materials. Results showed that the addition of SF and GGBS reduces the amount of micro-capillary pores (10 < d < 100 nm) and the total pore volume. The results indicate that the transport properties are related to the pore structure. The incorporation of SF reduced the permeability of the concrete by an order of magnitude. The pore distribution and pore composition had a significant effect on the gas permeability. The difference in porosity obtained using the MIP and NMR tests was large due to differences in testing techniques.
PubMed: 38541519
DOI: 10.3390/ma17061365