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.     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

Water as a complex system: its role in ligand diffusion, general anesthesia, and sleep

The work and inspiration of Robert Rosen is stated and expressed in personal tones. The concept of passages through water (H2O) near protein surfaces is reviewed in terms of its influence on ligand diffusion to an effector. This is offered as a target for interference by a non-specific general anesthetic agent. In view of the similarities between this anesthetic state and sleep, this mechanism is proposed to be operative for the sleep/wake states. Based on this mechanism and other factors, nitrogen (N2) is proposed as an exogenous sleep factor.     

Sequence analysis and rule development of predicting protein stability change upon mutation using decision tree model

Understanding the mechanism of the protein stability change is one of the most challenging tasks. Recently, the prediction of protein stability change affected by single point mutations has become an interesting topic in molecular biology. However, it is desirable to further acquire knowledge from large databases to provide new insights into the nature of them. This paper presents an interpretable prediction tree method (named iPTREE-2) that can accurately predict changes of protein stability upon mutations from sequence based information and analyze sequence characteristics from the viewpoint of composition and order. Therefore, iPTREE-2 based on a regression tree algorithm exhibits the ability of finding important factors and developing rules for the purpose of data mining. On a dataset of 1859 different single point mutations from thermodynamic database, ProTherm, iPTREE-2 yields a correlation coefficient of 0.70 between predicted and experimental values. In the task of data mining, detailed analysis of sequences reveals the possibility of the compositional specificity of residues in different ranges of stability change and implies the existence of certain patterns. As building rules, we found that the mutation residues in wild type and in mutant protein play an important role. The present study demonstrates that iPTREE-2 can serve the purpose of predicting protein stability change, especially when one requires more understandable knowledge.     

Long-time molecular dynamics simulations of botulinum biotoxin type-A at different pH values and temperatures

Botulinum neurotoxins type A (BoNT/A) are highly potent toxins, but are also useful in the treatment of illnesses. We studied the properties of BoNT/A at various temperatures and pH values in order to understand its toxicity and structure variations. The pH values of the environment of BoNT/A are obtained by changing the protonation states of certain titratable residue groups. Our results show that certain parts of the protein are active at acidic pH environments or at high temperatures. The protein is more stable in neutral environments at normal human body temperature, whereas, at high temperature, the protein is more stable in acidic environments. Also, the three domains of the protein tend to have relative motion rather than within individual domains.     

中国黍稷种质资源蛋白质和脂肪含量的鉴定分析

对来源于我国14省（区）的6515份黍稷种质资源,通过3次大批量的蛋白质和脂肪含量的鉴定分析,计算出黍稷蛋白质、脂肪的平均含量。筛选出一批蛋白质含量15．00％以上的高蛋白种质和脂肪含量4．00％以上的高脂肪种质,以及蛋白质含量15.00％以上、脂肪含量4．00％以上的双高种质,提供我国黍稷生产和育种利用。     

An unconventionally secreted effector from the root knot nematode Meloidogyne incognita,Mi-ISC-1, promotes parasitism by disrupting salicylic acid biosynthesis in host plants

Plant-parasitic nematodes need to deliver effectors that suppress host immunity for successful parasitism. We have characterized a novel isochorismatase effector from the root-knot nematode Meloidogyne incognita, named Mi-ISC-1. The Mi-isc-1 gene is expressed in the subventral oesophageal glands and is up-regulated in parasitic-stage juveniles. Tobacco rattle virus-induced gene silencing targeting Mi-isc-1 attenuated M. incognita parasitism. Enzyme activity assays confirmed that Mi-ISC-1 can catalyse hydrolysis of isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate in vitro. Although Mi-ISC-1 lacks a classical signal peptide for secretion at its N-terminus, a yeast invertase secretion assay showed that this protein can be secreted from eukaryotic cells. However, the subcellular localization and plasmolysis assay revealed that the unconventional secretory signal present on the Mi-ISC-1 is not recognized by the plant secretory pathway and that the effector was localized within the cytoplasm of plant cells, but not apoplast, when transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Ectopic expression of Mi-ISC-1 in N. benthamiana reduced expression of the PR1 gene and levels of salicylic acid (SA), and promoted infection by Phytophthora capsici. The cytoplasmic localization of Mi-ISC-1 is required for its function. Moreover, Mi-ISC-1 suppresses the production of SA following the reconstitution of the de novo SA biosynthesis via the isochorismate pathway in the cytoplasm of N. benthamiana leaves. These results demonstrate that M. incognita deploys a functional isochorismatase that suppresses SA-mediated plant defences by disrupting the isochorismate synthase pathway for SA biosynthesis to promote parasitism.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

Recent advances in the structural analysis of adenylation domains in natural product biosynthesis

Adenylation (A) domains catalyze the biosynthetic incorporation of acyl building blocks into nonribosomal peptides and related natural products by selectively transferring acyl substrates onto cognate carrier proteins (CP). The use of noncanonical acyl units, such as nonproteinogenic amino acids and keto acids, by A domains expands the structural diversity of natural products. Furthermore, interrupted A domains, which have embedded auxiliary domains, are able to modify the incorporated acyl units. Structural information on A domains is important for rational protein engineering to generate unnatural compounds. In this review, we summarize recent advances in the structural analysis of A domains. First, we discuss the mechanisms by which A domains recognize noncanonical acyl units. We then focus on the interactions of A domains with CP domains and embedded auxiliary domains.     

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Genome-wide physical protein–protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.