Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.     

Metabolomic analysis of pancreatic beta cells following exposure to high glucose

Background

Chronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects.

Methods

The BRIN-BD11 and INS-1E beta cell lines were cultured in 25 mM glucose for 20 h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, ¹H NMR and ¹³C NMR.

Results

Conditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident.

Conclusions

Results indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion.

General significance

Alterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell.     

Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1‐11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane‐spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O‐layer inserted in the outer membrane and the I‐layer inserted in the inner membrane. While the structure of the O‐layer has been solved by X‐ray crystallography, there is no detailed structural information on the I‐layer. Using high‐resolution cryo‐electron microscopy and molecular modelling combined with biochemical approaches, we determined the I‐layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.     

Unconventional protein secretion: an evolving mechanism

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.     

Diurnal Variation in the Symptoms of Colds and Influenza

The present research examined diurnal variation in the severity of symptoms of experimentally-induced colds and influenza. Nasal secretion was greatest in the morning, decreased over the day, and then showed a slight increase in the late evening. Colds did not change the average temperature, nor did they alter the temperature rhythm. Similar, negative results were found with alertness ratings.

Influenza B illnesses produced an increase in nasal secretion and systemic effects. The average temperature increased during this illness and subjects reported that they felt more drowsy. Diurnal variation in the severity of local and central symptoms was observed, with nasal secretion and the temperature increase being greatest in the early morning. These results have important implications for the assessment and treatment of the illnesses.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Analysis of the growth of Chlorella cultures using electronically measured cell-volume distribution data

Cell-number density and cell-volume distribution data were obtained from cultures of Chlorella fusca var. vacuolata Shihira & Krauss growing under both continuous and periodic illumination. Mean, median and modal cell-volumes were calculated from the cell-volume distributions and a high correlation shown between mean and median values, whilst mean and modal values did not correlate well. It was concluded that where the computation necessary for deriving mean cell-volumes was not practicable, the median cell-volume was the next most useful statistic.

Synchrony indices of discontinuously illuminated cultures gave similar values when based on change in cell-number and change in mean cell-volume. Cultures under continuous illumination showed synchronous divisions at the beginning of growth. These divisions gave high values of a synchrony index based on change in cell-number, but low values of an index based on change in mean cell-volume. It was concluded that mean cell-volume data are more sensitive to the occurrence of unbalanced growth than analysis of cell-number data.     

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (Review)

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.