Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.

During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28 ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits.

Preprotein signature for full susceptibility to the co-translational translocation inhibitor cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA.

Nanoparticles prepared from porcine cells support the healing of cutaneous inflammation in mice and wound re-epithelialization in human skin.

Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L.

Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis With Therapeutic Potential.

Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP).

Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL-p97 complex.

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS).

Inhibitors of Protein Translocation Across the ER Membrane.

Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast-growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors.

Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation.

Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes.

Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation.

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences.

ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7.

Background: The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role.

Results: We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export.

TRAP assists membrane protein topogenesis at the mammalian ER membrane.

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them.

Single rRNA helices bind independently to the protein-conducting channel SecYEG.

Ribosomes tightly interact with protein-conducting channels in the plasma membrane of bacteria (SecYEG) and in the endoplasmic reticulum of eukaryotes (Sec61 complex). This interaction is mediated by multiple junctions and is highly conserved during evolution. Although it is well known that both ribosomal proteins and ribosomal RNA (rRNA) are involved in the ribosome-channel interaction, detailed analyses on how these components contribute to this binding are lacking.

EcmPred: prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection.

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases.

Peroxisome formation requires the endoplasmic reticulum channel protein Sec61.

In peroxisome formation, models of near-autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec61 complex, the protein-conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec61 in yeast disables peroxisome formation.

BLProt: prediction of bioluminescent proteins based on support vector machine and relieff feature selection.

Background: Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances.

Prediction of apoptosis protein locations with genetic algorithms and support vector machines through a new mode of pseudo amino acid composition.

Apoptosis is an essential process for controlling tissue homeostasis by regulating a physiological balance between cell proliferation and cell death. The subcellular locations of proteins performing the cell death are determined by mostly independent cellular mechanisms. The regular bioinformatics tools to predict the subcellular locations of such apoptotic proteins do often fail.

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes.

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway.

A single Sec61-complex functions as a protein-conducting channel.

During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex.

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane.

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle.