The influence of protein folding on late stages of the secretion of α-amylases from Bacillus subtilis

A derivative of the α-amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme. This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane. When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca²⁺ ions for in vitro folding. When the concentration of Ca²⁺ in the growth medium was increased, so too did the relative yield of AmyLQS50.5. We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B. subtilis.     

The apyrase KlYnd1p of Kluyveromyces lactis affects glycosylation, secretion, and cell wall properties

The Kluyveromyces lactis ORF r_klactIV3,463 on chromosome IV, hereafter named KlYND1, encodes an endoapyrase that has nucleoside phosphatase activity with a lumenal orientation. The enzyme showed equally high activity towards GDP/UDP and ADP, and also showed activity, although to a lesser extent, towards GTP. No activity was detected with the other triphosphates and all monophosphates. The overexpression of KlYND1 in Klgda1Delta cells of K. lactis, devoid of the encoded GDPase/UDPase activity, suppressed the loss of O-glycosylation and cell wall-related defects described in such mutants, and suggests a partial overlap of function between the two genes, and therefore some redundancy. The overexpression of KlYND1 in wild-type cells enhanced the secretion of the recombinant human serum albumin and glucoamylase employed as reporters.     

Effects of divalent cations on phosphatase secretion in cultured tobacco cells

Some differences were found between Mg²⁺- and Ca²⁺-stimulated phosphatase secretion in cultured tobacco cells. The effect of Mg²⁺ ions was greater than that of Ca²⁺ ions, and Ca²⁺ ions at below 1 mM rather depressed the secretion. Upon the addition of Mg²⁺ ions plus Ca²⁺ ions, a synergistic stimulation of the secretion occurred. Different influences on the effects of Mg²⁺ and Ca²⁺ ions on the secretion were exerted by treating cells with metabolic inhibitors that reduced the level of cellular metabolic energy. Phosphate (Pi) and arsenate did not depress the secretion in the presence of Mg²⁺ ions, but did depress it in the presence of Ca²⁺ ions. These results strongly suggested that the secretion of phosphatase involved at least two different steps affected by divalent cations.     

Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungas Oidiodendron maius

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium. In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides. Some of these proteins were microsequenced and identified through homology search in databases. Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms. The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.     

Role of autophagy in unconventional protein secretion

In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.     

Role of autophagy in unconventional protein secretion

In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.     

枯草芽孢杆菌蛋白质分泌机制研究进展

综述了枯草芽孢杆菌不同蛋白质分泌机制,重点讨论了大多数细菌蛋白分泌的Sec途径,包括Sec途径的信号肽,信号肽酶,SecYEG通道,与分泌有关的各种细胞因子以及Sec途径的限制因素,此外还简要讨论了Tat途径,该途径能够转运折叠迅速或归密的蛋白质。     

Comparison of different signal peptides for protein secretion in nonlytic insect cell system

Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.     

革兰阴性杆菌对头孢哌酮／舒巴坦耐药率的变迁

目的调查革兰阴性杆菌对头孢哌酮／舒巴坦的耐药率变化。方法收集2004年1月至2007年12月从我院住院患者各种临床标本中分离的革兰阴性杆菌,使用VITEK-60全自动微生物分析仪进行菌种的鉴定,采用K-B法对头孢哌酮／舒巴坦进行药敏试验,对结果进行回顾性凋查。结果肠杆菌科细菌对头孢哌酮／舒巴坦的耐药率较低,大肠埃希菌、肺炎克雷伯菌和阴沟肠杆菌对头孢哌酮／舒巴坦的耐药率分别为1．7％,7．7％和5．8％。嗜麦芽窄食假单胞菌、洋葱伯克霍尔德菌、脑膜脓毒性金黄杆菌、铜绿假单胞菌和鲍曼不动杆菌对头孢哌酮／舒巴坦的耐药率分别为27．6％、38．0％、2．3％、23．5％和4．8％。铜绿假单胞菌和鲍曼不动杆菌对头孢哌酮／舒巴坦的耐药率从2004年的35．1％和23．7％下降至2007年的12．4％和0．4％。结论头孢哌酮／舒巴坦对肠杆菌科细菌具有非常强的体外抗菌活性,铜绿假单胞菌和鲍曼不动杆菌对头孢哌酮／舒巴坦的耐药率呈下降趋势。     

Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids

AIMS: To examine whether inactivation of the dlt operon and increased charge density of the wall enhances secretion of heterologous proteins in industrial strains of Bacillus licheniformis. METHODS AND RESULTS: The dltA gene of B. licheniformis was cloned, sequenced and mutated by inserting a chloramphenicol acetyl transferase (cat) gene cassette. The mutation facilitated growth in the late exponential growth phase, increased endogenous autolysis and decreased resistance to a cationic peptide, polylysine. It was observed that dltA mutation increased the production of cyclodextrin glycosyltransferase (CGTase) by 1.5- to sevenfold depending on the growth phase, but decreased the production of penicillinase by twofold. CONCLUSIONS AND SIGNIFICANCE: The results suggest that the d-alanylation of teichoic acids is an element that can be used to improve the production of some secretory proteins in industrial applications based on this important industrial microorganism.     

Role of calcium signaling in epithelial bicarbonate secretion

Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.     

Cyanobacterial leader peptides for protein secretion

The leader peptide of the major secreted protein PilA1 of the cyanobacterium Synechocystis sp. strain PCC 6803 and several artificial leader peptides have been used to study secretion of the reporter protein lichenase to the culture medium. The strains of Synechocystis carrying lichenase with the leader sequences of PilA and with the leader sequence of Slr2016 efficiently secreted the reporter protein. The artificial leader sequence that was characterized by the overall positive charge (as PilA1 and Slr2016 leaders) also allowed secretion. The artificial leader with negative charge, however, did not allow secretion of the reporter protein. Moreover, no secreted proteins have been isolated from this strain using conventional techniques for preparation of secreted proteins. These data suggest that the general secretion pathway in cyanobacteria, at least for pilins, recognizes the overall charge of the leader sequences, and operates in a sequence-non-specific manner.     

The wax glands and wax secretion of Matsucoccus matsumurae at different development stages

In this paper, the wax secretions and wax glands of Matsucoccus matsumurae (Kuwana) at different instars were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The first and second instar nymphs were found to secrete wax filaments via the wax glands located in the atrium of the abdominal spiracles, which have a center open and a series of outer ring pores. The wax gland of the abdominal spiracle possesses a large central wax reservoir and several wax-secreting cells. Third-instar male nymphs secreted long and translucent wax filaments from monolocular, biolocular, trilocular and quadrilocular pores to form twine into cocoons. The adult male secreted long and straight wax filaments in bundles from a group of 18–19 wax-secreting tubular ducts on the abdominal segment VII. Each tube duct contained five or six wax pores. The adult female has dorsal cicatrices distributed in rows, many biolocular tubular ducts and multilocular disc pores with 8–12 loculi secreting wax filaments that form the egg sac, and a rare type wax pores with 10 loculi secreting 10 straight, hollow wax filaments. The ultrastructure and cytological characteristics of the wax glands include wax-secreting cells with a large nucleus, multiple mitochondria and several rough endoplasmic reticulum. The functions of the wax glands and wax secretions are discussed.     

重症监护病房革兰阴性杆菌耐药性分析

目的了解深圳市人民医院重症监护病房(ICU)革兰阴性杆菌的分布及其耐药性,指导临床合理用药。方法收集来自重症监护病房各类标本分离的革兰阴性杆菌540株,用VITEK AMS-60或VITEK-Ⅱ全自动微生物分析仪进行菌种鉴定,用K-B法进行药敏试验。结果ICU检出的革兰阴性杆菌以鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主,ESBLs阳性的大肠埃希菌和肺炎克雷伯菌比例为61.6%和51.8%,各类细菌对常用抗菌药物表现为严重耐药和多重耐药。结论该院ICU检出的革兰阴性杆菌以鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主,且呈现多重耐药性。     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Experimental pitfalls in evaluating vectorial protein secretion in vitro; Sertoli cell secretion of androgen-binding protein and transferrin in two-compartment culture chambers

Summary We examined the influence of various Millipore filter pretreatments on the amounts of androgen-binding protein (ABP) and transferrin (Trf) found in the outer (OC) and inner (IC) compartment of two-compartment Sertoli cell (Sc) cultures. When Sc were cultured on untreated Millipore filters, less than 10% of ABP was found in OC during 3 initial culture days compared to similar cultures on pretreated filters. Most of the glycoprotein was shown to be bound by the filters. Pretreatment of Millipore filters with 5% bovine serum albumin (BSA) or 2% fetal bovine serum (FBS) maximally saturated the nonspecific protein-binding sites resulting in OC:IC ratio of ABP similar to that found in cultures on polycarbonate membranes, which exhibit very low protein-binding capacity. In contrast to ABP, about 40% of Trf was bound by the Millipore filter on Day 1, with only trace amounts bound thereafer. These differences were due to much higher secretion rate of Trf than ABP, resulting in a relatively smaller fraction of Trf bound to the filter. Again, the nonspecific binding of Trf was greatly reduced by filter pretreatment with 5% BSA or 2% FBS. It is concluded that complete saturation of protein-binding sites of cellulose ester supports is necessary for reliable evaluation of vectorial protein secretion by Sc and other polarized epithelial cells maintained in this type of culture. The implications of partial saturation of protein-binding sites of culture support in interpreting experimental results are discussed. This work was supported in part by grant HD-17802 (A.S.) from the National Institutes of Health, Bethesda, MD.     

Dynamic modeling of cooperative protein secretion in microorganism populations

Interactions between microorganisms can have a crucial effect on their population dynamics. Typically, interactions are mediated through the environment by molecules and proteins that are products of cell metabolism and physiology; they therefore reflect the internal dynamics of the single cell. In this work we aim to integrate single-cell properties of gene expression that affect indirect interactions between microorganisms under challenging conditions, into a quantitative model of population dynamics. Specifically we address the problem of a microbial population secreting a protein that can actively extract a growth-limiting resource, such as a simple sugar or iron, from the environment. The genes coding for the protein can undergo random epigenetic transitions between active and silenced states, and can be repressed by the product of their reaction. We model cooperative and competitive interactions between protein producing and non-producing phenotypes by nonlinear dynamical systems and analyze them both in terms of asymptotic states and of transient dynamics. Our model shows that phenotypic transitions allow a stable coexistence of the two phenotypes, and enables us to make predictions regarding the conditions required for such coexistence and the typical timescales of transient dynamics. It also shows how repression by the reaction product induces a feedback at the population-environment level that can result in limit cycle dynamics. The relation of these results to experiments are discussed.     

Regulation of Wnt protein secretion and its role in gradient formation

In metazoans, many developmental and disease-related processes are mediated by Wnt proteins, which are secreted by specific cells to regulate cellular programmes in the surrounding tissue. Although the Wnt-induced signal-transduction cascades are well studied, little is known about how Wnts are secreted. The discovery of Porcupine, an endoplasmic-reticulum-resident acyltransferase, led to closer inspection of the secretory routes of Wnts, and the analysis of Wnt secretion has become an exciting new area of research. Wnt post-translational modifications, interaction partners and subcellular localizations now indicate that Wnt release is tightly regulated. In this review, we summarize recent advances in the field of Wnt secretion and discuss the possibility that separate pathways might regulate the release of lipid-linked morphogens for short-range and long-range signalling.