Mycobacteria, such as the pathogen , utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transp...Mycobacteria, such as the pathogen , utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.
DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEA...DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1-RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1-RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.
Cancer cell fusion represents a rare event. However, the surviving cancer hybrid cells after a post-hybrid selection process (PHSP) can overgrow other cancer cells by exhibiting a proliferation advantage and/or expressio...Cancer cell fusion represents a rare event. However, the surviving cancer hybrid cells after a post-hybrid selection process (PHSP) can overgrow other cancer cells by exhibiting a proliferation advantage and/or expression of cancer stem-like properties. Addition of new tumor properties during hetero-fusion of cancer cells e.g. with mesenchymal stroma-/stem-like cells (MSC) contribute to enhanced tumor plasticity via acquisition of new/altered functionalities. This provides new avenues for tumor development and metastatic behavior. Consequently, the present review article will also address the question as to whether cancer cell fusion represents a general and possibly evolutionary-conserved program or rather a random process?
Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information flow between neurons and understanding their dynam...Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information flow between neurons and understanding their dynamics is the key to unravel their role in behavior. To understand how the brain transmits information and how brain states arise, it is essential to visualize the dynamics of neurotransmitters, neuromodulators and neurochemicals. In the last five years, an increasing number of single-wavelength biosensors either based on periplasmic binding proteins (PBPs) or on G-protein-coupled receptors (GPCR) have been published that are able to detect neurotransmitter release and with high spatial and temporal resolution. Here we review and discuss recent progress in the development of these sensors, their limitations and future directions.
During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering...During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation of RNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.
Soluble nuclear transport receptors and stationary nucleoporins are at the heart of the nucleocytoplasmic transport machinery. A subset of nucleoporins contains characteristic and repetitive FG (phenylalanine-glycine) mo...Soluble nuclear transport receptors and stationary nucleoporins are at the heart of the nucleocytoplasmic transport machinery. A subset of nucleoporins contains characteristic and repetitive FG (phenylalanine-glycine) motifs, which are the basis for the permeability barrier of the nuclear pore complex (NPC) that controls transport of macromolecules between the nucleus and the cytoplasm. FG-motifs can interact with each other and/or with transport receptors, mediating their translocation across the NPC. The molecular details of homotypic and heterotypic FG-interactions have been analyzed at the structural level. In this review, we focus on the interactions of nucleoporins with nuclear transport receptors. Besides the conventional FG-motifs as interaction spots, a thorough structural analysis led us to identify additional similar motifs at the binding interface between nucleoporins and transport receptors. A detailed analysis of all known human nucleoporins revealed a large number of such phenylalanine-containing motifs that are not buried in the predicted 3D-structure of the respective protein but constitute part of the solvent-accessible surface area. Only nucleoporins that are rich in conventional FG-repeats are also enriched for these motifs. This additional layer of potential low-affinity binding sites on nucleoporins for transport receptors may have a strong impact on the interaction of transport complexes with the nuclear pore and, thus, the efficiency of nucleocytoplasmic transport.
The essential ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of G...The essential ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of = 0.49 ± 0.28 s and = 0.014 ± 0.003 s, respectively. We further verified these rates with photocaged NPEAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis ( < 2 × 10 s). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.
This study attempted to investigate the effect of circ_0051799 on the immune microenvironment of lung adenocarcinoma (LUAD) and the relationship between circ_0051799 and exosomes. The number and morphology of exosomes we...This study attempted to investigate the effect of circ_0051799 on the immune microenvironment of lung adenocarcinoma (LUAD) and the relationship between circ_0051799 and exosomes. The number and morphology of exosomes were verified by nanoparticle tracking, transmission electron microscopy and western blotting. CCK8, EdU, Transwell and flow cytometry were used to verify the regulatory role of exosomes and circ_0051799 on tumor progression. Dual luciferase reporting and RNA immunoprecipitation were used to verify the targeted regulatory relationship between circ_0051799, miR-214-3p and IGF2BP3. WB was used to verify the role of the JAK/STAT pathway in circ_0051799 regulation. Ectopic tumor grafts and models were used to validate their role in regulating LUAD progression. Hypoxic environment could alter but does not alter its shape. Exosomes can participate in the regulation of macrophage polarization by circ_0051799. and assays had shown that circ_0051799 could affect the proliferation and metastasis of LUAD through targeting miR-214-3p mediated IGF2BP3 regulated JAK/STAT pathway. This study found that hypoxia can affect LUAD process by promoting the regulation of macrophage polarization by exosome circ_0051799.
Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via...Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.
Atg18, Atg21 and Hsv2 are homologous β-propeller proteins binding to PI3P and PI(3,5)P. Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with bo...Atg18, Atg21 and Hsv2 are homologous β-propeller proteins binding to PI3P and PI(3,5)P. Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with both the ER and the vacuole. Atg21 is restricted to the vacuole phagophore contact, where it organizes part of the Atg8-lipidation machinery. The role of Hsv2 is less understood, it partly affects micronucleophagy. Atg18 is further involved in regulation of PI(3,5)P synthesis. Recently, a novel Atg18-retromer complex and its role in vacuole homeostasis and membrane fission was uncovered.
Na/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol ) and is responsible for the sodium-dependent uptake of bile salts across the basolateral memb...Na/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol ) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.
Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete st...Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete structures of both yeast and human Mediator complexes and have a better understanding of their interactions with the Pol II pre-initiation complex (PIC). Herein, we provide a summary of recent achievements and discuss their implications for future studies of Mediator and its role in gene regulation.
In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of p...In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection.
The cytoskeleton of eukaryotes consists of actin filaments, microtubules and intermediate filaments (IF). IFs, in particular, are prone to pronounced phosphorylation, leading to additional charges on the affected amino a...The cytoskeleton of eukaryotes consists of actin filaments, microtubules and intermediate filaments (IF). IFs, in particular, are prone to pronounced phosphorylation, leading to additional charges on the affected amino acids. In recent years, a variety of experiments employing either reconstituted protein systems or living cells have revealed that these altered charge patterns form the basis for a number of very diverse cellular functions and processes, including reversible filament assembly, filament softening, network remodeling, cell migration, interactions with other protein structures, and biochemical signaling.
Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation and which is in principle conserved throughout the kingdoms of life is the N-degron pathway, f...Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation and which is in principle conserved throughout the kingdoms of life is the N-degron pathway, formerly called the 'N-end rule'. In the cytosol of eukaryotes and prokaryotes, N-terminal residues can be major determinants of protein stability. While the eukaryotic N-degron pathway depends on the ubiquitin proteasome system, the prokaryotic counterpart is driven by the Clp protease system. Plant chloroplasts also contain such a protease network, which suggests that they might harbor an organelle specific N-degron pathway similar to the prokaryotic one. Recent discoveries indicate that the N-terminal region of proteins affects their stability in chloroplasts and provides support for a Clp-mediated entry point in an N-degron pathway in plastids. This review discusses structure, function and specificity of the chloroplast Clp system, outlines experimental approaches to test for an N-degron pathway in chloroplasts, relates these aspects into general plastid proteostasis and highlights the importance of an understanding of plastid protein turnover.
The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH) into ATP, the cell's energy currency in a process named oxidative phospho...The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII + CIV). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P -module is downregulated and the P -module is completely abolished in NDUFB10 knock outs.
Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thr...Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbβ, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.
Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that per...Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein-protein and protein-lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.