Assessment of AlphaFold2 for Human Proteins via Residue Solvent Exposure.

As only 35% of human proteins feature (often partial) PDB structures, the protein structure prediction tool AlphaFold2 (AF2) could have massive impact on human biology and medicine fields, making independent benchmarks of interest. We studied AF2's ability to describe the backbone solvent exposure as a functionally important and easily interpretable "natural coordinate" of protein conformation, using human proteins as test case. After screening for appropriate comparative sets, we matched 1818 human proteins predicted by AF2 against 7585 unique experimental PDBs, and after curation for sequence overlap, we assessed 1264 comparative pairs comprising 115 unique AF2 structures and 652 unique experimental structures. AF2 performed markedly worse for multimers, whereas ligands, cofactors, and experimental resolution were interestingly not very important for performance. AF2 performed excellently for monomer proteins. Challenges relating to specific groups of residues and multimers were analyzed. We identified larger deviations for lower-confidence scores (pLDDT), and exposed residues and polar residues (e.g., Asp, Glu, Asn) being less accurately described than hydrophobic residues. Proline conformations were the hardest to predict, probably due to a common location in dynamic solvent-accessible parts. In summary, using solvent exposure as a metric, we quantified the performance of AF2 for human proteins and provided estimates of the expected agreement as a function of ligand presence, multimer/monomer status, local residue solvent exposure, pLDDT, and amino acid type. Overall performance was found to be excellent.

Survival of the cheapest: how proteome cost minimization drives evolution.

Darwin's theory of evolution emphasized that positive selection of functional proficiency provides the fitness that ultimately determines the structure of life, a view that has dominated biochemical thinking of enzymes as perfectly optimized for their specific functions. The 20th-century modern synthesis, structural biology, and the central dogma explained the machinery of evolution, and nearly neutral theory explained how selection competes with random fixation dynamics that produce molecular clocks essential e.g. for dating evolutionary histories. However, quantitative proteomics revealed that selection pressures not relating to optimal function play much larger roles than previously thought, acting perhaps most importantly via protein expression levels. This paper first summarizes recent progress in the 21st century toward recovering this universal selection pressure. Then, the paper argues that proteome cost minimization is the dominant, underlying 'non-function' selection pressure controlling most of the evolution of already functionally adapted living systems. A theory of proteome cost minimization is described and argued to have consequences for understanding evolutionary trade-offs, aging, cancer, and neurodegenerative protein-misfolding diseases.

A model of proteostatic energy cost and its use in analysis of proteome trends and sequence evolution.

A model of proteome-associated chemical energetic costs of cells is derived from protein-turnover kinetics and protein folding. Minimization of the proteostatic maintenance cost can explain a range of trends of proteomes and combines both protein function, stability, size, proteostatic cost, temperature, resource availability, and turnover rates in one simple framework. We then explore the ansatz that the chemical energy remaining after proteostatic maintenance is available for reproduction (or cell division) and thus, proportional to organism fitness. Selection for lower proteostatic costs is then shown to be significant vs. typical effective population sizes of yeast. The model explains and quantifies evolutionary conservation of highly abundant proteins as arising both from functional mutations and from changes in other properties such as stability, cost, or turnover rates. We show that typical hypomorphic mutations can be selected against due to increased cost of compensatory protein expression (both in the mutated gene and in related genes, i.e. epistasis) rather than compromised function itself, although this compensation depends on the protein's importance. Such mutations exhibit larger selective disadvantage in abundant, large, synthetically costly, and/or short-lived proteins. Selection against increased turnover costs of less stable proteins rather than misfolding toxicity per se can explain equilibrium protein stability distributions, in agreement with recent findings in E. coli. The proteostatic selection pressure is stronger at low metabolic rates (i.e. scarce environments) and in hot habitats, explaining proteome adaptations towards rough environments as a question of energy. The model may also explain several trade-offs observed in protein evolution and suggests how protein properties can coevolve to maintain low proteostatic cost.

Computational chemistry of modified [MFe3S4] and [M2Fe2S4] clusters: assessment of trends in electronic structure and properties.

The aim of this work is to understand the molecular evolution of iron-sulfur clusters in terms of electronic structure and function. Metal-substituted models of biological [Fe(4)S(4)] clusters in oxidation states [M(x)Fe(4-x)S(4)](3+/2+/1+) have been studied by density functional theory (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, and Pd, with x = 1 or 2). Most of these clusters have not been characterized before. For those that have been characterized experimentally, very good agreement is obtained, implying that also the predicted structures and properties of new clusters are accurate. Mean absolute errors are 0.024 A for bond lengths ([Fe(4)S(4)], [NiFe(3)S(4)], [CoFe(3)S(4)]) and 0.09 V for shifts in reduction potentials relative to the [Fe(4)S(4)] cluster. All structures form cuboidal geometries similar to the all-iron clusters, except the Pd-substituted clusters, which instead form highly distorted trigonal and tetragonal local sites in compromised, pseudocuboidal geometries. In contrast to other electron-transfer sites, cytochromes, blue copper proteins, and smaller iron-sulfur clusters, we find that the [Fe(4)S(4)] clusters are very insensitive to metal substitution, displaying quite small changes in reorganization energies and reduction potentials upon substitution. Thus, the [Fe(4)S(4)] clusters have an evolutionary advantage in being robust to pollution from other metals, still retaining function. We analyze in detail the electronic structure of individual clusters and rationalize spin couplings and redox activity. Often, several configurations are very close in energy, implying possible use as spin-crossover systems, and spin states are predicted accurately in all but one case ([CuFe(3)S(4)]). The results are anticipated to be helpful in defining new molecular systems with catalytic and magnetic properties.

Improved interaction potentials for charged residues in proteins.

Electrostatic interactions dominate the structure and free energy of biomolecules. To obtain accurate free energies involving charged groups from molecular simulations, OPLS-AA parameters have been reoptimized using Monte Carlo free energy perturbation. New parameters fit a self-consistent, experimental set of hydration free energies for acetate (Asp), propionate (Glu), 4-methylimidazolium (Hip), n-butylammonium (Lys), and n-propylguanidinium (Arg), all resembling charged residue side chains, including beta-carbons. It is shown that OPLS-AA free energies depend critically on the type of water model, TIP4P or TIP3P; i.e., each water model requires specific water-charged molecule interaction potentials. New models (models 1 and 3) are thus described for both water models. Uncertainties in relative free energies of charged residues are approximately 2 kcal/mol with the new parameters, due to variations in system setup (MAEs of ca. 1 kcal/mol) and noise from simulations (ca. 1 kcal/mol). The latter error of approximately 1 kcal/mol contrasts MAEs from standard OPLS-AA of up to 13 kcal/mol for the entire series of charged residues or up to 5 kcal/mol for the cationic series Lys, Arg, and Hip. The new parameters can be used directly in molecular simulations with no modification of neutral residues needed and are envisioned to be particular important in simulations where charged residues change environment.

A cost-effectiveness analysis of immunotherapy with SQ allergen extract for patients with seasonal allergic rhinoconjunctivitis in selected European countries.

BACKGROUND: Seasonal allergic rhinoconjunctivitis can, for some people, reduce quality of life and the ability to cope with everyday tasks. SCOPE: In this paper we investigate the cost-effectiveness of immunization therapy with Alutard SQ (ASQ) and compare the cost-effectiveness in countries where the therapy has been in use in order to assess the impact of national therapeutic practices on the results of health economic assessments. Data are obtained from a clinical trial carried out in 2001-2002. To evaluate the cost-effectiveness of immunization we have added data on resource use in Austria, Denmark, Finland, Germany, The Netherlands, and Sweden. FINDINGS: The computations result in cost-effectiveness ratios for allergen immunization between 10,000 euros and 20,000 euros per QALY even without provision for indirect costs, and achieving dominance in most countries where indirect costs have also been taken into account. The country comparisons show that the direct cost of administrating the up-dosing and maintenance differs considerably between countries, and that the cost of medical staff is substantial, constituting in most cases more than half of the direct costs of the immunization therapy. CONCLUSION: The study shows that immunotherapy with SQ allergen extract is cost-effective in a wide range of national environments, and that cost-effectiveness differences by country are largely a result of different practices in the up-dosing phase.

[In vitro methods for phototoxicity and photocarcinogenicity testing of drugs]. / In vitro Methoden zur Prüfung von Arzneimitteln auf photogenotoxische/photokanzerogene Eigenschaften.

Phototoxicity is an acknowledged property of some UV and/or visible light absorbing substances some of which are used as pharmaceuticals or in cosmetic preparations. In recent years attention has been called upon the fact that toxic intermediates that are generated upon photoactivation of a substance can also lead to DNA damage. Such damage may lead to mutated/initiated skin cells which in turn can contribute to an elevated skin cancer risk. The method of choice to test for photo-related skin carcinogenesis is a 1-year study in genetically hairless mice in which the formation of skin papilloma and their latency time are assessed. Here, in vitro test approaches to test for photogenotoxicity can be used in a tiered assessment approach asking the use of in vitro genotoxicity tests for prediction of rodent/human carcinogenicity. In the past few years some effort has been put into the evaluation for such systems, in particular standard test protocols have been generated for the in vitro photo-micronucleus test and the in vitro photo-comet assay with Chinese hamster V79 cells. The data that have been produced so far show promising results regarding the implementation of these systems in a tiered approach for photocarcinogenicity assessment of UV- and/or visible light absorbing substances but the systems will have to be validated in further collaborative studies.

Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals.

Issues of biological relevance and thresholds for genotoxicity are discussed here based upon the background of experience with the submissions for the approval of new pharmaceuticals to the German regulatory authority over the period between 1990 and 1997. This experience shows that out of the genotoxicity test systems which are required according to existing guidelines in the European Union (EU), the in vitro tests for chromosomal aberrations (CA) and the mouse lymphoma tk assays (MLA) yield a rate of positives that is about four-fold higher than that of other genotoxicity tests. A detailed analysis of chemical and pharmacological classes of compounds and their effects in these systems reveals that in addition to direct DNA reactivity several mechanisms of indirect genotoxicity such as nucleoside analogue incorporation into DNA, interaction with microtubule assembly, topoisomerase inhibition and high levels of cytotoxicity are relevant. New pharmaceuticals, for which the latter mechanisms apply, often display threshold-like characteristics in their genotoxic effects in vitro or even in vivo in experimental animals. This casts doubt upon the relevance of positive in vitro test results for such compounds. However, the discussion of examples shows that it may not be easy to demonstrate the exact thresholded mechanism of genotoxicity in a given case. In particular, the demonstration of a coincidence of genotoxicity and high levels of cytotoxicity, which seems to be a major factor for biologically non-relevant in vitro positive new pharmaceuticals, usually requires quite extensive testing. Hence, for new pharmaceuticals it is practice to provide in addition to in vitro results that may be thresholded a wealth of information from in vivo studies on genotoxicity, carcinogenicity, metabolism, pharmacokinetics, etc. the results of which help in assessing the biological relevance of in vitro positives. The regulatory acknowledgement of biologically non-relevant, thresholded mechanisms of (in vitro) genotoxicity in addition to those that are considered relevant for human risk ensures a better understanding of test results and is needed for the credibility of genotoxicity testing practice in general.

An assessment of the in vitro hepatocyte micronucleus assay.

The in vitro hepatocyte micronucleus assay was tested for its practicability and its usefulness in detecting mutagens. The assay protocol developed by Alati et al. (1989) was shown to give reproducible levels of proliferating hepatocytes and the formation of micronuclei could be readily assessed by fluorescence microscopy. Epidermal growth factor and insulin were used as mitogens, yielding mitotic indices of 2.4 +/- 0.74% after 72 h of culture. The high number of 8.0 +/- 3.33% micronucleated hepatocytes in control cultures at that time, typically for in vitro stimulated hepatocytes, is probably due to disordered mitoses frequently leading to chromosome loss. The direct acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine and the clastogens cyclophosphamide and retrorsine, which require metabolic activation, induced dose dependent increases in the frequencies of micronucleated hepatocytes. The carcinogen 2-AAF also yielded significantly enhanced rates of micronuclei. The non-mutagen KCl as well as the peroxisome proliferator clofibrate, which is considered to be a non-genotoxic hepatocarcinogen, yielded consistently negative results. Problems occurred when chemicals exerting strong cytotoxic effects were tested in this assay. The mutagen and hepatocarcinogen aflatoxin B1 did not enhance the number of micronucleated hepatocytes. Rather a reduction of micronuclei and of mitoses was observed at AFB1 concentrations considered positive in other genotoxicity assays. Hepatocyte proliferation seems to be highly susceptible to the cytotoxic action of chemicals. A decrease in the proliferating activity of hepatocytes can obviously prevent the detection of mutagenic effects. Further studies on the in vitro hepatocyte micronucleus assay are necessary to clarify its role in mutagenicity testing.