Analysis of yeast prion aggregates with amyloid-staining compound in vivo

Yeast prions are protein-based genetic elements whose non-Mendelian patterns of inheritance are explained by their inheritance of altered conformations. Here we showed that aggregates made by overexpression of two different prion domains of Sup35 and Rnq1, were stained in yeast by thioflavin-S, an amyloid binding compound. These results suggested that yeast prion domains take the form of amyloid in vivo, and supported the idea that the self-propagating property of amyloids is responsible for the heritable traits of yeast prions.     

Illuminating intracellular signaling and molecules for single cell analysis

Fluorescent and bioluminescent proteins are now widely used for detection of small molecules and various intracellular events ranging from protein conformational change to cell death in living cells. To analyze the dynamics of molecular processes in real time at the level of single cells, engineered protein-based probes with higher sensitivity and selectivity are required. The probes can be entirely genetically encoded and can comprise fusions of different proteins or domains. This review specifically examines basic concepts of designing genetically encoded fluorescent and bioluminescent probes developed in the past decade, highlighting some potential applications for basic research and for drug discovery.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Cloning strategy for producing brush-forming protein-based polymers

Brush-forming polymers are being used in a variety of applications, and by using recombinant DNA technology, there exists the potential to produce protein-based polymers that incorporate unique structures and functions in these brush layers. Despite this potential, production of protein-based brush-forming polymers is not routinely performed. For the design and production of new protein-based polymers with optimal brush-forming properties, it would be desirable to have a cloning strategy that allows an iterative approach wherein the protein based-polymer product can be produced and evaluated, and then if necessary, it can be sequentially modified in a controlled manner to obtain optimal surface density and brush extension. In this work, we report on the development of a cloning strategy intended for the production of protein-based brush-forming polymers. This strategy is based on the assembly of modules of DNA that encode for blocks of protein-based polymers into a commercially available expression vector; there is no need for custom-modified vectors and no need for intermediate cloning vectors. Additionally, because the design of new protein-based biopolymers can be an iterative process, our method enables sequential modification of a protein-based polymer product. With at least 21 bacterial expression vectors and 11 yeast expression vectors compatible with this strategy, there are a number of options available for production of protein-based polymers. It is our intent that this strategy will aid in advancing the production of protein-based brush-forming polymers.     

Crystal structure of a bacterial cocaine esterase.

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.     

Computational methods to predict protein aggregation

In most cases, protein aggregation stems from the establishment of non-native intermolecular contacts. The formation of insoluble protein aggregates is associated with many human diseases and is a major bottleneck for the industrial production of protein-based therapeutics. Strikingly, fibrillar aggregates are naturally exploited for structural scaffolding or to generate molecular switches and can be artificially engineered to build up multi-functional nanomaterials. Thus, there is a high interest in rationalizing and forecasting protein aggregation. Here, we review the available computational toolbox to predict protein aggregation propensities, identify sequential or structural aggregation-prone regions, evaluate the impact of mutations on aggregation or recognize prion-like domains. We discuss the strengths and limitations of these algorithms and how they can evolve in the next future.     

Protein-lipid interactions in membrane trafficking at the Golgi complex

The integrated interplay between proteins and lipids drives many key cellular processes, such as signal transduction, cytoskeleton remodelling and membrane trafficking. The last of these, membrane trafficking, has the Golgi complex as its central station. Not only does this organelle orchestrates the biosynthesis, transport and intracellular distribution of many proteins and lipids, but also its own function and structure is dictated by intimate functional and physical relationships between protein-based and lipid-based machineries. These machineries are involved in the control of the fundamental events that govern membrane traffic, such as in the budding, fission and fusion of transport intermediates, in the regulation of the shape and geometry of the Golgi membranes themselves, and, finally, in the generation of "signals" that can have local actions in the secretory system, or that may affect other cellular systems. Lipid-protein interactions rely on the abilities of certain protein domains to recognize specific lipids. These interactions are mediated, in particular, through the headgroups of the phospholipids, although a few of these protein domains are able to specifically interact with the phospholipid acyl chains. Recent evidence also indicates that some proteins and/or protein domains are more sensitive to the physical environment of the membrane bilayer (such as its curvature) than to its chemical composition.     

Protein-based hybrid catalysts--design and evolution

Artificial metalloenzymes result from the introduction of a catalytically competent non-native metal cofactor within a protein environment. In the present contribution, we summarize the recent achievements in the design and the optimization of such protein-based hybrid catalysts, with an emphasis on enantioselective transformations. The second part outlines the milestones required to achieve en masse production, screening and directed evolution of artificial metalloenzymes. In the spirit of Darwinian evolution, this will allow the full potential of such protein-based hybrid catalysts to be fully unraveled, thus complementing both homogeneous and enzymatic catalysis.     

Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins

Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of proteins in living cells. Although the technique is relatively old, its application to studying endogenous intracellular proteins in living cells is relatively recent and is a consequence of the newly developed fluorescent protein-based living cell protein tags. This is particularly true for nuclear proteins, in which endogenous protein mobility has only recently been studied. Here we examine the experimental design and analysis of FRAP experiments. Mathematical modeling of FRAP data enables the experimentalist to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion coefficient of the molecule under study. As experimentalists begin to dissect the relative influence of protein domains within individual proteins, this approach will allow a quantitative assessment of the relative influences of different molecular interactions on the steady-state distribution and protein function in vivo.     

Rnq1: an epigenetic modifier of protein function in yeast

Two protein-based genetic elements (prions) have been identified in yeast. It is not clear whether other prions exist, nor is it understood how one might find them. We established criteria for searching protein databases for prion candidates and found several. The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble. The insoluble state is dominant and transmitted between cells through the cytoplasm. When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion [PSI+], the phenotypic and epigenetic behavior of [PSI+] was fully recapitulated. These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.     

Elastic molecular machines in metabolism and soft-tissue restoration.

Elastic protein-based machines (bioelastic materials) can be designed to perform diverse biological energy conversions. Coupled with the remarkable energy-conversion capacity of cells, this makes possible a tissue-restoration approach to tissue engineering. When properly attached to the extracellular matrix, cells sense the forces to which they are subjected and respond by producing an extracellular matrix that will withstand those forces. Elastic protein-based polymers can be designed as temporary functional scaffoldings that cells can enter, attach to, spread, sense forces and remodel, with the potential to restore natural tissue.     

Protein-based natural antibacterial materials and their applications in food preservation

Plastics materials used for food packaging are recalcitrant, leading to a growing global environmental problem, which arouses the attention of environmental protection departments in many countries. Therefore, to meet the increasing demand for sustainable and environment-friendly consumer products, it is necessary for the food industry to develop natural antibacterial materials for food preservation. This review summarizes the common biodegradable natural antimicrobial agents and their applications in food preservation; as well as an overview of five commonly used biodegradable protein-based polymers, such as zein, soy protein isolate, gelatin and whey protein, with special emphasis on the advantages of protein-based biopolymers and their applications in food packaging industry.     

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions—thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.     

<Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis> (DSM 20083) and <Emphasis Type="Italic">Lactobacillus casei</Emphasis> (Lafti L26-DSL): Probiotics Able to Block the In Vitro Adherence of Rotavirus in MA104 Cells

Rotavirus is the leading worldwide cause of gastroenteritis in children under five years of age. Even though there are some available vaccines to prevent the disease, there are limited strategies for challenging diarrhea induced by rotavirus infection. For this reason, researchers are constantly searching for other approaches to control diarrhea by means of probiotics. In order to demonstrate the ability of some probiotic bacteria to interfere with the in vitro rotavirus infection in MA104 cells, strains of Lactobacillus sp. and Bifidobacterium sp. were tested in MA104 cells before the viral infection. As a preliminary assay, a blocking effect treatment was performed with viable bacteria. In this screening assay, four of initial ten bacteria showed a slight reduction of the viral infection (measured by percentage of infection). L. casei (Lafti L26-DSL), L. fermentum(ATCC 9338), B. adolescentis (DSM 20083), and B. bifidum (ATCC 11863) were used in further experiments. Three different treatments were tested in order to evaluate protein-based metabolites obtained from mentioned bacteria: (i) cell exposure to the protein-based metabolites before viral infection, (ii) exposure to protein-based metabolites after viral infection, and (iii) co-incubation of the virus and protein-based metabolites before viral infection to the cell culture. The best effect performed by protein-based metabolites was observed during the co-incubation assay of the virus and protein-based metabolites before adding them into the cell culture. The results showed 25 and 37% of infection in the presence of L. casei and B. adolescentis respectively. These results suggest that the antiviral effect may be occurring directly with the viral particle instead of making a blocking effect of the cellular receptors that are needed for the viral entrance.     

Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture

Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods.     

Three approaches to assembling nano-bio-machines using molecular motors

Efforts to use protein molecular motors as nanoactuators are making rapid progress. For instance, it is now possible to carry out directional transport of small cargo along microtracks or microchannels using kinesin-microtubule systems, which could be the basis of micro-conveyor belts or molecular shuttles. However, the applicability of protein-based devices is limited by their poor stability in artificial environments. In addition, assembly of complex, intelligent microdevices or systems will likely require bottom-up self-assembly, and we still do not have sufficient knowledge to rationally design self-assembling protein-based microdevices or systems. One approach to solving the problems associated with protein-based systems is to use DNA-based nanodevices, which are amenable to rational design. Indeed, ingenious design has enabled realization of DNA-based nanoactuators and self-assembled micropatterns of various shapes. One also could use cells, organelles, or tissues as preassembled motile units, and several motile devices have already been realized using this approach. In addition to being less prone to the assemaly problems, cell-based microdevices have the advantage that the motile units reproduce themselves, and genetically encoded functional modifications can be replicated effortlessly. These protein-based, DNA-based, and cell-based systems each have distinct advantages and disadvantages, so that hybrid devices combining the best characteristics of all three would seem the most likely to succeed.     

Biomimetic design of nanopatterned membranes

The biomimetic approach copying the supramolecular building principle of many archaeal cell envelopes (i.e., a plasma membrane with associated S-layer proteins) has resulted in stable lipid membranes with excellent reconstitution properties for transmembrane proteins. This is a particular challenge as one-third of all proteins in an organism are membrane proteins like pores, ion channels, or receptors. At S-layer supported lipid membranes, spatial well-defined domains on the S-layer protein interact noncovalently with lipid head groups within the lipid membrane resulting in a nanopatterning of a few anchored and scores of diffusional free-lipid molecules. In addition, no impact on the hydrophobic core region and on the function of reconstituted integral proteins has been determined. Among others, particularly S-layer stabilized membranes can be used for structure-function studies on reconstituted integral proteins and also in the membrane protein-based molecular nanotechnology, e.g., in the design of biosensing devices (e.g., lipid chip or lab-on-a-chip), or for receptor or ion channel-based high-throughput screening.     

Assigning function to yeast proteins by integration of technologies

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.