Structure of the CLC-1 chloride channel from Homo sapiens.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters

Bacterial protein translocation requires only one copy of the SecY complex in vivo

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation

Structure of the substrate-engaged SecA-SecY protein translocation machine.

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecAs two-helix finger is close to the polypeptide, while SecAs clamp interacts with the polypeptide in a sequence-independent manner by inducing a short β-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel

Do we need to reconsider the CMAM admission and discharge criteria?; an analysis of CMAM data in South Sudan

Background: Weight-for-height Z-score (WHZ) and Mid Upper Arm Circumference (MUAC) are both commonly used as acute malnutrition screening criteria. However, there exists disparity between the groups identified as malnourished by them. Thus, here we aim to investigate the clinical features and linkage with chronicity of the acute malnutrition cases identified by either WHZ or MUAC. Besides, there exists evidence indicating that fat restoration is disproportionately rapid compared to that of muscle gain in hospitalized malnourished children but related research at community level is lacking. In this study we suggest proxy measure to inspect body composition restoration responding to malnutrition management among the malnourished children. Methods: The data of this study is from World Vision South Sudan’s emergency nutrition program from 2006 to 2012 (4443 children) and the nutrition survey conducted in 2014 (3367 children). The study investigated clinical presentations of each type of severe acute malnutrition (SAM) by WHZ (SAM-WHZ) or MUAC (SAM-MUAC), and analysed correlation between each malnutrition and chronic malnutrition. Furthermore, we explored the pattern of body composition restoration during the recovery phase by comparing the relative velocity of MUAC3 with that of weight gain. Results: As acutely malnourished children identified by MUAC more often share clinical features related to chronic malnutrition and minimal overlapping with malnourished children by WHZ, Therefore, MUAC only screening in the nutrition program would result in delayed identification of the malnourished children. Conclusions: The relative velocity of MUAC3 gain was suggested as a proxy measure for volume increase, and it was more prominent than that of weight gain among the children with SAM by WHZ and MUAC over all the restoring period. Based on this we made a conjecture about dominant fat mass gain over the period of CMAM program. Also, considering initial weight gain could be ascribed to fat mass increase, the current discharge criteria would leave the malnourished children at risk of mortality even after treatment due to limited restoration of muscle mass. Given this, further research should be followed including assessment of body composition for evidence to recapitulate and reconsider the current admission and discharge criteria for CMAM program

Structure of the SecY channel during initiation of protein translocation

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel

4-O-Carboxymethylascochlorin Inhibits Expression Levels of on Inflammation-Related Cytokines and Matrix Metalloproteinase-9 Through NF–κB/MAPK/TLR4 Signaling Pathway in LPS-Activated RAW264.7 Cells

Toll-like receptor 4 (TLR4) and matrix metalloproteinase-9 (MMP-9) are known to play important roles in inflammatory diseases such as arteriosclerosis and plaque instability. The purpose of this study was to perform the effect of 4-O-carboxymethylascochlorin (AS-6) on MMP-9 expression in lipopolysaccharide (LPS)-induced murine macrophages and signaling pathway involved in its anti-inflammatory effect. Effect of AS-6 on MAPK/NF-κB/TLR4 signaling pathway in LPS-activated murine macrophages was examined using ELISA, Western blotting, reverse transcription polymerase chain reaction (RT-PCR) and fluorescence immunoassay. MMP-9 enzyme activity was examined by gelatin zymography. AS-6 significantly suppressed MMP-9 and MAPK/NF-κB expression levels in LPS-stimulated murine macrophages. Expression levels of inducible nitric oxide synthase (iNOS), COX2, MMP-9, JNK, ERK, p38 phosphorylation, and NF-κB stimulated by LPS were also decreased by AS-6. Moreover, AS-6 suppressed TLR4 expression and dysregulated LPS-induced activators of transcription signaling pathway. The results of this study showed that AS-6 can inhibit LPS-stimulated inflammatory response by suppressing TLR4/MAPK/NF-κB signals, suggesting that AS-6 can be used to induce the stability of atherosclerotic plaque and prevent inflammatory diseases in an in vitro model

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain

Mechanistic Studies of SecY-Mediated Protein Translocation in Intact Escherichia coli Cells

During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain