Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.     

BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid–Liquid Phase Separation

The liquid–liquid phase separation (LLPS) of biomolecules is a phenomenon which is nowadays recognized as the driving force for the biogenesis of numerous functional membraneless organelles and cellular bodies. The interplay between the protein primary sequence and phase separation remains poorly understood, despite intensive research. To uncover the sequence-encoded signals of protein capable of undergoing LLPS, we developed a novel web platform named BIAPSS (Bioinformatics Analysis of LLPS Sequences). This web server provides on-the-fly analysis, visualization, and interpretation of the physicochemical and structural features for the superset of curated LLPS proteins.     

The Effects of Mutations on Protein Function: A Comparative Study of Three Databases of Mutations in Humans

Single-nucleotide mutations (SNPs) in protein-coding regions of the human genome are a major factor in determining human variation in health and disease. Here, we analyze the amino acid changes and functional effects due to non-synonymous SNPs. Three databases were used: (i) Variation – mutations found in the general human population; (ii) Cosmic – mutations found in cancer cells; and (iii) Pathogenic – a curated subset of mutations in Variation that are associated with diseases. The distributions of amino acid changes in these datasets were analyzed. It is shown that mutations in the Pathogenic dataset, in particular, tend to introduce order-promoting residues. The effects of the mutations in these datasets were also studied using the program Polyphen-2, which predicts the functional impact of non-synonymous mutations. In order to evaluate the significance of these predicted effects, we compared them to those due to the same amino acid replacements introduced at other positions in the same proteins as a control. A mutation can be deleterious because the amino acid change is drastic (for example a change from hydrophobic residue to hydrophilic residue) or because of its location in the protein. We found that, on both counts, mutations in the Variation dataset tend to be less deleterious than randomly expected whereas mutations in the Pathogenic dataset tend to be more deleterious than their control mutations. The mutations in the Cosmic dataset are found to be more deleterious than those in its control set but less than those in Pathogenic.     

Arrow of Time,Entropy, and Protein Folding: Holistic View on Biochirality

Chirality is a universal phenomenon, embracing the space–time domains of non-organic and organic nature. The biological time arrow, evident in the aging of proteins and organisms, should be linked to the prevalent biomolecular chirality. This hypothesis drives our exploration of protein aging, in relation to the biological aging of an organism. Recent advances in the chirality discrimination methods and theoretical considerations of the non-equilibrium thermodynamics clarify the fundamental issues, concerning the biphasic, alternative, and stepwise changes in the conformational entropy associated with protein folding. Living cells represent open, non-equilibrium, self-organizing, and dissipative systems. The non-equilibrium thermodynamics of cell biology are determined by utilizing the energy stored, transferred, and released, via adenosine triphosphate (ATP). At the protein level, the synthesis of a homochiral polypeptide chain of L-amino acids (L-AAs) represents the first state in the evolution of the dynamic non-equilibrium state of the system. At the next step the non-equilibrium state of a protein-centric system is supported and amended by a broad set of posttranslational modifications (PTMs). The enzymatic phosphorylation, being the most abundant and ATP-driven form of PTMs, illustrates the principal significance of the energy-coupling, in maintaining and reshaping the system. However, the physiological functions of phosphorylation are under the permanent risk of being compromised by spontaneous racemization. Therefore, the major distinct steps in protein-centric aging include the biosynthesis of a polypeptide chain, protein folding assisted by the system of PTMs, and age-dependent spontaneous protein racemization and degradation. To the best of our knowledge, we are the first to pay attention to the biphasic, alternative, and stepwise changes in the conformational entropy of protein folding. The broader view on protein folding, including the impact of spontaneous racemization, will help in the goal-oriented experimental design in the field of chiral proteomics.     

Small Heat Shock Protein 22 Improves Cognition and Learning in the Tauopathic Brain

The microtubule-associated protein tau pathologically accumulates and aggregates in Alzheimer’s disease (AD) and other tauopathies, leading to cognitive dysfunction and neuronal loss. Molecular chaperones, like small heat-shock proteins (sHsps), can help deter the accumulation of misfolded proteins, such as tau. Here, we tested the hypothesis that the overexpression of wild-type Hsp22 (wtHsp22) and its phosphomimetic (S24,57D) Hsp22 mutant (mtHsp22) could slow tau accumulation and preserve memory in a murine model of tauopathy, rTg4510. Our results show that Hsp22 protected against deficits in synaptic plasticity and cognition in the tauopathic brain. However, we did not detect a significant change in tau phosphorylation or levels in these mice. This led us to hypothesize that the functional benefit was realized through the restoration of dysfunctional pathways in hippocampi of tau transgenic mice since no significant benefit was measured in non-transgenic mice expressing wtHsp22 or mtHsp22. To identify these pathways, we performed mass spectrometry of tissue lysates from the injection site. Overall, our data reveal that Hsp22 overexpression in neurons promotes synaptic plasticity by regulating canonical pathways and upstream regulators that have been characterized as potential AD markers and synaptogenesis regulators, like EIF4E and NFKBIA.     

Retro-MoRFs: Identifying Protein Binding Sites by Normal and Reverse Alignment and Intrinsic Disorder Prediction

Many cell functions in all living organisms rely on protein-based molecular recognition involving disorder-to-order transitions upon binding by molecular recognition features (MoRFs). A well accepted computational tool for identifying likely protein-protein interactions is sequence alignment. In this paper, we propose the combination of sequence alignment and disorder prediction as a tool to improve the confidence of identifying MoRF-based protein-protein interactions. The method of reverse sequence alignment is also rationalized here as a novel approach for finding additional interaction regions, leading to the concept of a retro-MoRF, which has the reversed sequence of an identified MoRF. The set of retro-MoRF binding partners likely overlap the partner-sets of the originally identified MoRFs. The high abundance of MoRF-containing intrinsically disordered proteins in nature suggests the possibility that the number of retro-MoRFs could likewise be very high. This hypothesis provides new grounds for exploring the mysteries of protein-protein interaction networks at the genome level.     

Circularly permuted dihydrofolate reductase of E.coli has functional activity and a destabilized tertiary structure

Three circularly permuted variants of Escherichia coli dihydrofolatereductase genes were constructed. Linkers coding tri- and pentapeptideswere used to connect the natural 5'- and 3'-terminal ends. Onlyone variant of circularly permuted protein with tripeptide linkerand the cleavage of the peptide bond between 107 and 108 aminoacid residues was produced in a good yield. The expressed proteinwas insoluble in the cells, but at pH 8.0 and higher the isolatedprotein was soluble. Enzymatic assay and physical studies haveshown that permuted dihydrofolate reductase has a destabilizedtertiary structure. Only the addition of the natural substratesor Inhibitors lead to the protein with the native-like structureand functional activity.     

The Finite Size Effects and Two-State Paradigm of Protein Folding

The coil to globule transition of the polypeptide chain is the physical phenomenon behind the folding of globular proteins. Globular proteins with a single domain usually consist of about 30 to 100 amino acid residues, and this finite size extends the transition interval of the coil-globule phase transition. Based on the pedantic derivation of the two-state model, we introduce the number of amino acid residues of a polypeptide chain as a parameter in the expressions for two cooperativity measures and reveal their physical significance. We conclude that the k2 measure, defined as the ratio of van ’t Hoff and calorimetric enthalpy is related to the degeneracy of the denatured state and describes the number of cooperative units involved in the transition; additionally, it is found that the widely discussed k2=1 is just the necessary condition to classify the protein as the two-state folder. We also find that Ωc, a quantity not limited from above and growing with system size, is simply proportional to the square of the transition interval. This fact allows us to perform the classical size scaling analysis of the coil-globule phase transition. Moreover, these two measures are shown to describe different characteristics of protein folding.