New information on the waste-disposal units of living cells

"Using electron microscopy and a revolutionary new system for protein expression, we have determined at a subnanometer scale the complete architecture, including the relative positions of all its protein components, of the proteasome regulatory particle," says biophysicist Eva Nogales, the research team's co-principal investigator. "This provides a structural basis for the ability of the proteasome to recognize and degrade unwanted proteins and thereby regulate the amount of any one type of protein that is present in the cell."

Says the team's other co-principal investigator and corresponding author, biochemist Andreas Martin, "While the biochemical function of many of the proteasome components have been determined, and some subnanometer structures have been identified, it was unclear before now which component goes where and which components interact with one another. Now we have a much better understanding as to how the proteasome machinery works to control cellular processes and this opens the possibility of manipulating proteasome activity for the treatment of cancer and other diseases."

Nogales, who holds appointments with Berkeley Lab, UC Berkeley, and the Howard Hughes Medical Institute, and Martin, who holds appointments with UC Berkeley and the QB3 Institute, are the senior authors of a paper describing this work in the journal Nature. The paper is titled "Complete subunit architecture of the proteasome regulatory particle." Other co-authors were Gabriel Lander, Eric Estrin, Mary Matyskiela and Charlene Bashore.

At any given moment, a human cell typically contains about 100,000 different proteins, with certain proteins being manufactured and others being discarded as needed for the cell's continued prosperity. Unwanted proteins are tagged with a "kiss-of-death" label in the form of a polypeptide called "ubiquitin." A protein marked with ubiquitin is delivered to any one of the some 30,000 proteasomes in the cell – barrel-shaped complexes which act as waste disposal units that rapidly break-down or degrade the protein. The 2004 Nobel Prize in chemistry was awarded to a trio of scientists who first described the proteasome process, but a lack of structural information has limited the scientific understanding of the mechanics behind this process.

Nogales, an expert on electron microscopy and image analysis, and Martin, who developed the new protein expression system used in this work, combined the expertise of their respective research groups to study the proteasome regulatory particle in yeast. The particle features 19 sub-units that are organized into two sub-complexes, a "lid" and a "base." The lid contains the regulatory elements that identify the ubiquitin tag marking a protein for destruction, and the base features a hexameric ring that pulls the tagged protein inside the chamber of the proteasome barrel where it is degraded.

"The lid consists of nine non-ATPase proteins including ubiquitin receptors that accept properly tagged proteins but prevent a protein not marked for degradation from engaging with the proteasome," Nogales says. "Since degradation is irreversible, it is critical that only ubiquitin-tagged proteins engage the proteasome. Interestingly, the ubiquitin tag has to be removed before the protein can be translocated into the proteasome's destruction chamber, so the lid also contains de-ubiquitination enzymes that remove the tags after the protein has engaged with the proteasome."

The proteasome regulatory particle's base contains six distinct AAA+ ATPases that form the hetero-hexameric ring, which serves as the molecular motor of the proteasome.

"We predict that the ATPases use the energy of ATP binding and hydrolysis to exert a pulling force on engaged proteins, unfolding and translocating them through a narrow central pore and into the degradation chamber," Martin says. "The steps in the proteasome process – from protein recognition to de-ubiquitination and degradation have to be very highly coordinated in time and space. Locating all of these components and identifying their relative orientations has been very telling about how the processes are coordinated with each other."

Nogales credits the protein expression system developed by Martin and his research group, in which proteins are expressed and assembled in bacteria, as being critical to the success of this research.

"Until now researchers had to work with purified protein complexes from the cell, which could not be manipulated or modified in any way," she says. "Andy Martin's new heterologous expression system allows for the manipulation and dissection of protein functions. For our studies it was crucial to generate lid sub-complexes that had one marker at a time in each of the subunits so that we could determine the position of each protein within the lid. With this new system we generated truncations, deletions and fusion constructs that were used to localize individual subunits and delineate their boundaries within the lid."

Provided by Lawrence Berkeley National Laboratory (news : web)

12 new flavonoids discovered in Kew tree

Analysis and identification

Geoffrey Kite analysed an extract of the leaves of C. kentukea and found several of the flavonoids known from S. japonicum, but the overall mixture of flavonoids in C. kentukea was much more complicated, consisting of more than 50 compounds. Emily Rowe, a student from the University of Bath who was working at Kew for a year as part of her degree course, was given the task of trying to separate some of the flavonoids from the mixture so that their structures could be determined. She obtained 13 examples, whose structures were elucidated using a technique called nuclear magnetic resonance spectroscopy (NMR) by Nigel Veitch, who realised that 12 of them were new to science. The structures of these compounds were reported recently in the scientific journal Phytochemistry.

With this knowledge, Geoffrey Kite was able to suggest probable structures for another 39 of the flavonoids in the leaf extract using a technique called liquid chromatography-mass spectrometry. Many of these are probably new to science as well, but it was not possible to prove this without first purifying them and determining their structures by NMR.

Cladrastis kentukea at Kew

Cladrastis kentukea is a medium-sized tree endemic to eastern North America. The hard, close-grained wood is clear yellow when first cut and was used by early American pioneers to make gunstocks and furniture. Although it is not common in the wild, the species is widely grown for its ornamental value. There are several specimens of C. kentukea growing at Kew together with two other Cladrastis species, C. sinensis and C. platycarpa. Most can be found in the ‘legume dell’ near to the Pavilion Restaurant. One specimen of C. sinensis is growing in the corner of the outside seating area of the restaurant, where visitors can enjoy a meal in the shade of a new scientific discovery, since this species also contains many of the new flavonoids.

More information: Kite, G. C., et al.(2011). Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae). Phytochemistry 72: 372-384.

Provided by Royal Botanic Gardens, Kew

Why coffee drinking reduces the risk of Type 2 diabetes

Ling Zheng, Kun Huang and colleagues explain that previous studies show that coffee drinkers are at a lower risk for developing Type 2 diabetes, which accounts for 90-95 percent of diabetes cases in the world. Those studies show that people who drink four or more cups of coffee daily have a 50 percent lower risk of Type 2 diabetes. And every additional cup of coffee brings another decrease in risk of almost 7 percent. Scientists have implicated the misfolding of a substance called human islet amyloid polypeptide (hIAPP) in causing Type 2 diabetes, and some are seeking ways to block that process. Zheng and Huang decided to see if coffee's beneficial effects might be due to substances that block hIAPP.

Indeed, they identified two categories of compounds in coffee that significantly inhibited hIAPP. They suggest that this effect explains why coffee drinkers show a lower risk for developing diabetes. "A beneficial effect may thus be expected for a regular coffee drinker," the researchers conclude.

More information: Coffee Components Inhibit Amyloid Formation of Human Islet Amyloid Polypeptide in Vitro: Possible Link between Coffee Consumption and Diabetes Mellitus, J. Agric. Food Chem., 2011, 59 (24), pp 13147–13155. DOI: 10.1021/jf201702h

Abstract
Global epidemic studies have suggested that coffee consumption is reversely correlated with the incidence of type 2 diabetes mellitus (T2DM), a metabolic disease. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Coffee extracts have three major active components: caffeine, caffeic acid (CA), and chlorogenic acid (CGA). In this study, the effects of these major coffee components, as well as dihydrocaffeic acid (DHCA) (a major metabolite of CGA and CA), on the amyloidogenicity of hIAPP were investigated by thioflavin-T based fluorescence emission, transmission electronic microscopy, circular dichroism, light-induced cross-linking, dynamic light scattering, and MTT-based cell viability assays. The results suggest that all components show varied inhibitory effects on the formation of toxic hIAPP amyloids, in which CA shows the highest potency in delaying the conformational transition of the hIAPP molecule with the most prolonged lag time, whereas caffeine shows the lowest potency. At a 5-fold excess molar ratio of compound to hIAPP, all coffee-derived compounds affect the secondary structures of incubated hIAPP as suggested by the circular dichroism spectra and CDPro deconvolution analysis. Further photoinduced cross-linking based oligomerization and dynamic light scattering studies suggested CA and CGA significantly suppressed the formation of hIAPP oligomers, whereas caffeine showed no significant effect on oligomerization. Cell protection effects were also observed for all three compounds, with the protection efficiency being greatest for CA and least for CGA. These findings suggest that the beneficial effects of coffee consumption on T2DM may be partly due to the ability of the major coffee components and metabolites to inhibit the toxic aggregation of hIAPP.

Provided by American Chemical Society (news : web)

Chemical engineers boost petrochemical output from biomass by 40 percent

"We think that today we can be economically competitive with crude oil production," says research team leader George Huber, an associate professor of chemical engineering at UMass Amherst and one of the country's leading experts on catalytic pyrolysis.

Huber says his research team can take wood, grasses or other renewable biomass and create five of the six petrochemicals that serve as the building blocks for the chemical industry. They are benzene, toluene, and xylene, which are aromatics, and ethylene and propylene, which are olefins. Methanol is the only one of those six key petrochemicals not produced in that same single-step reaction.

"The ultimate significance of our research is that products of our green process can be used to make virtually all the petrochemical materials you can find. In addition, some of them can be blended into gasoline, diesel or jet fuel," says Huber.

The new process was outlined in a paper published in the Dec. 23, 2011 edition of the German Chemical Society's journal Angewandte Chemie. It was written by Huber, Wei Fan, assistant professor of chemical engineering, and graduate students Yu-Ting Cheng, Jungho Jae and Jian Shi.

"The whole name of the game is yield," says Huber. "The question is what amount of aromatics and olefins can be made from a given amount of biomass. Our paper demonstrates that with this new gallium-zeolite catalyst we can increase the yield of those products by 40 percent. This gets us much closer to the goal of catalytic fast pyrolysis being economically viable. And we can do it all in a renewable way."

The new production process has the potential to reduce or eliminate industry's reliance on fossil fuels to make industrial chemicals worth an estimated $400 billion annually, Huber says. The team's catalytic fast pyrolysis technology has been licensed to New York City's Anellotech, Inc., co-founded by Huber, which is scaling up the process to industrial size for introduction into the petrochemical industry.

In this single-step catalytic fast pyrolysis process, either wood, agricultural wastes, fast growing energy crops or other non-food biomass is fed into a fluidized-bed reactor, where this feedstock pyrolysizes, or decomposes due to heating, to form vapors. These biomass vapors then enter the team's new gallium-zeolite (Ga-ZSM-5) catalyst, inside the same reactor, which converts vapors into the aromatics and olefins. The economic advantages of the new process are that the reaction chemistry occurs in one single reactor, the process uses an inexpensive catalyst and that aromatics and olefins are produced that can be used easily in the existing petrochemical infrastructure.

Olefins and aromatics are the building blocks for a wide range of materials. Olefins are used in plastics, resins, fibers, elastomers, lubricants, synthetic rubber, gels and other industrial chemicals. Aromatics are used for making dyes, polyurethanes, plastics, synthetic fibers and more.

Provided by University of Massachusetts at Amherst

Backing out of the nanotunnel: New method for nucleic acid analysis

In the world of biomolecules such as proteins and the hereditary nucleic acids DNA and RNA, three-dimensional structure determines function. Analysis of the passage of such molecules through nanopores offers a relatively new, but highly promising, technique for obtaining information about their spatial conformations. However, interactions between the test molecules and the proteins used as pores have so far hindered quantitative analysis of the behavior of even simply structured molecules within nanopores. This problem must be solved before the technique can be routinely used for structure determination. In a project carried out under the auspices of the Cluster of Excellence "Nanosystems Initiative Munich" (NIM), researchers led by LMU physicist Professor Ulrich Gerland and Professor Friedrich Simmel (Technical University of Munich) have developed a new method that depends on the analysis of reverse translocation through asymmetric pores, which minimizes the interference caused by interactions with the pore material itself. This approach has enabled the team to construct a theoretical model that allows them to predict the translocation dynamics of nucleic acids that differ in their nucleotide sequences.

The nucleic acids RNA and DNA both belong to the class of molecules known chemically as polynucleotides. Both are made up of strings of four basic types of building blocks called nucleotides, which fall into two complementary pairs. In their single-stranded forms, DNA and RNA can fold into what are called secondary structures, as complementary nucleotides in the sequence pair up, forcing the intervening segments to form loops. If the single-stranded loop is very short, the secondary structure is referred to as a hairpin. As in the case of proteins, the secondary structures of nucleic acids influence their biochemical functions. The elucidation of the secondary structure of nucleic acid sequences is therefore of great interest.

"Nanopores are increasingly being employed to investigate the secondary structures of RNA and DNA," Gerland points out. "Passage through narrow nanopores causes the sequence to unfold, and the dynamics of translocation provide insights into the structural features of the molecules, without the need to modify them by adding a fluorescent label. The technique is relatively new, and its potential has not yet been fully explored."

In the new study, he and his collaborators used a new experimental procedure, which allowed them to quantitatively describe the passage of simply structured polynucleotide sequences through nanopores, and develop a theoretical model that accounts for their findings. This level of understanding has not been achieved previously, because complicating factors such as interactions between the protein nanopore and the polynucleotide have had a significant influence on the measurements and made it difficult to predict the behavior of the test molecules.

Thanks to a clever change in experimental design, the impact of these factors has now been minimized. The trick is to perform the measurements on molecules as they translocate through the pore in reverse. First, the polynucleotide of interest is forced through the conical orifice from one side under the influence of an electrical potential. This causes its secondary structure to unfold and, as it emerges, the molecule refolds. An anchor at the end of the polynucleotide chain prevents it from passing completely through the pore onto the other side. For the return journey the potential is reversed, so that the process of unfolding now begins at the narrow end of the pore, and at this point the analysis is initiated.

"In contrast to the situation during forward translocation, no significant interactions appear to take place during the reverse trip," says Simmel.

On the basis of their experimental measurements, the researchers went on to construct a theoretical model that enabled them to predict the translocation dynamics of various hairpin structures with the aid of thermodynamic calculations of so-called "free-energy landscapes".

"This model could in the future provide the foundation of a procedure for the elucidation of the secondary structures of complex polynucleotides," says Gerland.

More information: "Quantitative Analysis of the Nanopore Translocation Dynamics of Simple Structured Polynucleotides" S. Schink, S. Renner, K. Alim, V. Arnaut, F.C. Simmel, U. Gerland Biophysical Journal Vol. 102, January 2012, pp 1-11. doi: 10.1016/j.bpj.2011.11.4011

Provided by Ludwig-Maximilians-Universitat Munchen