Effect of Gel Exposition on Calcium and Carbonate Ions Determines the Stm-l Effect on the Crystal Morphology of Calcium Carbonate.

Biomineralization of fish otoliths is regulated by macromolecules, such as proteins, whose presence is crucial for the functionality and properties of these mineralized structures. Special regulatory effects are exerted by intrinsically disordered proteins, such as the polyanionic Starmaker-like protein from medaka, a homolog of zebrafish Starmaker. In this study, we employed a set of bioinspired mineralization experiments with a single diffusion system to investigate the effect of the Starmaker-like protein on calcium carbonate biominerals with regards to the prior exposition of the protein to calcium or carbonate ions. Interestingly, the bioinspired minerals grown in the presence of the Starmaker-like protein in calcium- or carbonate-type experiments differ significantly in terms of morphology and protein distribution within the crystals. Our deeper analysis shows that the Starmaker-like protein action is a result of the environmental conditions to which it is exposed. These findings may be of special interest in the areas of biomineralization process pathways and biomaterial sciences.

Nesfatin-3 possesses divalent metal ion binding properties, which remain hidden in the nucleobindin-2 precursor protein.

BACKGROUND: Nucleobindin-2 (Nucb2) is a multidomain protein that, due to its structure, participates in many physiological processes. It was originally identified in several regions of the hypothalamus. However, more recent studies have redefined and extended the function of Nucb2 far beyond its initially observed role as a negative modulator of food intake. RESULTS: Previously, we described Nucb2 as structurally divided into two parts: the Zn2+-sensitive N-terminal half and the Ca2+-sensitive C-terminal half. Here, we investigated the structural and biochemical properties of its C-terminal half, which, after posttranslational processing, yields the formation of a fully uncharacterized peptide product known as nesfatin-3. Nesfatin-3 likely contains all the key respective structural regions of Nucb2. Hence, we expected that its molecular properties and affinity toward divalent metal ions might resemble those of Nucb2. Surprisingly, the obtained results showed that the molecular properties of nesftain-3 were completely different from those of its precursor protein. Moreover, we designed our work as a comparative analysis of two nesfatin-3 homologs. We noticed that in their apo forms, both proteins had similar shapes and existed in solution as extended molecules. They both interacted with divalent metal ions, and this interaction manifested itself in a compaction of the protein molecules. Despite their similarities, the differences between the homologous nesfatin-3s were even more informative. Each of them favored interaction with a different metal cation and displayed unique binding affinities compared either to each other or to Nucb2. CONCLUSIONS: The observed alterations suggested different from Nucb2 physiological roles of nesfatin-3 and different impacts on the functioning of the tissues and on metabolism and its control. Our results clearly demonstrated that nesfatin-3 possessed divalent metal ion binding properties, which remained hidden in the nucleobindin-2 precursor protein.

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures.

In this paper, we show that a hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is a rational method for the design of novel self-assembled nanomaterials based on peptides. Incorporation of a trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue into the outer positions of the model coiled-coil peptide led to the formation of helical foldamers, which was determined by circular dichroism (CD) and vibrational spectroscopy. The oligomerization state of the obtained peptides in water was established by analytical ultracentrifugation (AUC). The thioflavin T assay and Congo red methods showed that the obtained α,ß-peptides possess a strong tendency to aggregate, leading to the formation of self-assembled nanostructures, which were assessed by microscopic techniques. The location of the ß-amino acid in the heptad repeat of the coiled-coil structure proved to have an influence on the secondary structure of the obtained peptides and on the morphology of the self-assembled nanostructures.

Phase separation propensity of the intrinsically disordered AB region of human RXRß.

RXRß is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes - RXRα and RXRγ - RXRß has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRß (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid-liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR - RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR. Video Abstract.

Otolin-1, an otolith- and otoconia-related protein, controls calcium carbonate bioinspired mineralization.

BACKGROUND: Otoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents. METHODS: In this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy. RESULTS: We show that both proteins are embedded in the core of CaCO3 crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface. GENERAL SIGNIFICANCE: The results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.

Does one plus one always equal two? Structural differences between nesfatin-1, -2, and nesfatin-1/2.

Nesfatin-1 and -2 are produced from a reaction in which the N-terminus of human Nucleobindin-2 undergoes proteolytical processing. To date, Nucleobindin-2 and/or nesfatin-1 have only been shown to act as peptide hormones. On the other hand, the purpose of nesfatin-2 remains unknown. Since Nucleobindin-2/nesfatin-1 is thought impact the control of a wide range of physiological processes, including energy homeostasis, neurodegenerative processes and carcinogenesis, its ligands/interactions deserve special studies and attention. However, there are no reports about the molecular properties of the proteolytical products of human Nucleobindin-2 in the literature. Hence, this study aimed to analyze the effect of Zn(II) and Ca(II) on human nesfatin-1, -2, and -1/2 structures. Herein, we report that human nesfatin-1 is a member of the intrinsically disordered protein family, as indicated by circular dichroism and analytical ultracentrifugation experiments. In contrast, we found that the human nesfatin-2 and nesfatin-1/2 structures were globular with intrinsically disordered regions. Under Zn(II) treatment, we observed concentration-dependent structurization and compaction of intrinsically disordered nesfatin-1 and its propensity for oligomerization, as well as destabilization of both nesfatin-2 and nesfatin-1/2. Furthermore, dissociation constants for Zn(II) binding by nesfatin-1, nesfatin-2, and nesfatin-1/2 were also reported. Moreover, structurally distinct nesfatin-1 and -2 seem to be interdependent when linked together, as indicated by the observed molecular properties of nesfatin-1/2, which in turn are not a simple sum of the properties exhibited by the former peptides. Thus, herein, we shed new light on the molecular behavior of human nesfatins, which might help to elucidate the complex function of those peptides. Video abstract.

The Role of Intrinsically Disordered Proteins in Liquid-Liquid Phase Separation during Calcium Carbonate Biomineralization.

Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid-liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS.

Deep blue autofluorescence reflects the oxidation state of human transthyretin.

Human transthyretin (TTR) is a tetrameric protein transporting thyroid hormones and retinol. TTR is a neuroprotective factor and sensor of oxidative stress which stability is diminished due to mutations and aging, leading to amyloid deposition. Adverse environmental conditions, such as redox and metal ion imbalances, induce destabilization of the TTR structure. We have previously shown that the stability of TTR was disturbed by Ca2+ and other factors, including DTT, and led to the formation of an intrinsic fluorophore(s) emitting blue light, termed deep blue autofluorescence (dbAF). Here, we show that the redox state of TTR affects the formation dynamics and properties of dbAF. Free thiols lead to highly unstable subpopulations of TTR and the frequent ocurrence of dbAF. Oxidative conditions counteracted the destabilizing effects of free thiols to some extent. However, strong oxidative conditions led to modifications of TTR, which altered the stability of TTR and resulted in unique dbAF spectra. Riboflavin and/or riboflavin photoproducts bound to TTR and crosslinked TTR subunits. Riboflavin-sensitized photooxidation increased TTR unfolding, while photooxidation, either in the absence or presence of riboflavin, increased proteolysis and resulted in multiple oxidative modifications and dityrosine formation in TTR molecules. Therefore, oxidation can switch the role of TTR from a protective to pathogenic factor.

N'-terminal- and Ca2+-induced stabilization of high-order oligomers of full-length Danio rerio and Homo sapiens otolin-1.

Otolin-1 is a C1q family member and a major component of the organic matrix of fish otoliths and human otoconia. To date, the protein molecular properties have not been characterized. In this work, we describe biochemical characterization and comparative studies on saccular-specific otolin-1 derived from Danio rerio and Homo sapiens. Due to the low abundance of proteins in the otoconial matrix, we developed a production and purification method for both recombinant homologues of otolin-1. Danio rerio and Homo sapiens otolin-1 forms higher-order oligomers that can be partially disrupted under reducing conditions. The presence of Ca2+ stabilizes the oligomers and significantly increases the thermal stability of the proteins. Despite the high sequence coverage, the oligomerization of Danio rerio otolin-1 is more affected by the reducing conditions and presence of Ca2+ than the human homologue. The results show differences in molecular behaviour, which may be reflected in Danio rerio and Homo sapiens otolin-1 role in otolith and otoconia formation.

Controlling the conformational stability of coiled-coil peptides with a single stereogenic center of a peripheral ß-amino acid residue.

The key issue in the research on foldamers remains the understanding of the relationship between the monomers structure and conformational properties at the oligomer level. In peptidomimetic foldamers, the main goal of which is to mimic the structure of proteins, a main challenge is still better understanding of the folding of peptides and the factors that influence their conformational stability. We probed the impact of the modification of the peptide periphery with trans- and cis-2-aminocyclopentanecarboxylic acid (ACPC) on the structure and stability of the model coiled-coil using circular dichroism (CD), analytical ultracentrifugation (AUC) and two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Although, trans-ACPC and cis-ACPC-containing mutants differ by only one peripheral stereogenic center, their conformational stability is strikingly different.

Transcription Regulators and Membraneless Organelles Challenges to Investigate Them.

Eukaryotic cells are composed of different bio-macromolecules that are divided into compartments called organelles providing optimal microenvironments for many cellular processes. A specific type of organelles is membraneless organelles. They are formed via a process called liquid-liquid phase separation that is driven by weak multivalent interactions between particular bio-macromolecules. In this review, we gather crucial information regarding different classes of transcription regulators with the propensity to undergo liquid-liquid phase separation and stress the role of intrinsically disordered regions in this phenomenon. We also discuss recently developed experimental systems for studying formation and properties of membraneless organelles.

Deep blue autofluorescence reveals the instability of human transthyretin.

Wild-type human transthyretin (TTR) is a tetrameric protein that transports thyroxine and retinol in the blood and brain. However, a number of mutations or aging leads to destabilization of the quaternary structure of TTR, which results in dissociation of TTR tetramers to monomers, followed by oligomerization and subsequent amyloid formation. TTR amyloid is a pathogenic factor underlying several diseases. It has recently been documented that destabilization of the structure of TTR is driven by Ca2+. The present work shows that the in vitro redox conditions contribute to the destabilization and formation of the highly unstable substoichiometric population(s) of TTR molecules. Importantly, destabilized TTR forms acquire the ability to emit fluorescence in the blue range of the light spectrum. Dithiothreitol (DTT), in the presence of Ca2+, enhances the formation of complex autofluorophore which displays maxima at 417 nm and 438 nm in the emission spectrum of TTR.

Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1.

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure-function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

Transthyretin: From Structural Stability to Osteoarticular and Cardiovascular Diseases.

Transthyretin (TTR) is a tetrameric protein transporting hormones in the plasma and brain, which has many other activities that have not been fully acknowledged. TTR is a positive indicator of nutrition status and is negatively correlated with inflammation. TTR is a neuroprotective and oxidative-stress-suppressing factor. The TTR structure is destabilized by mutations, oxidative modifications, aging, proteolysis, and metal cations, including Ca2+. Destabilized TTR molecules form amyloid deposits, resulting in senile and familial amyloidopathies. This review links structural stability of TTR with the environmental factors, particularly oxidative stress and Ca2+, and the processes involved in the pathogenesis of TTR-related diseases. The roles of TTR in biomineralization, calcification, and osteoarticular and cardiovascular diseases are broadly discussed. The association of TTR-related diseases and vascular and ligament tissue calcification with TTR levels and TTR structure is presented. It is indicated that unaggregated TTR and TTR amyloid are bound by vicious cycles, and that TTR may have an as yet undetermined role(s) at the crossroads of calcification, blood coagulation, and immune response.

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Nucleobindin-2 consists of two structural components: The Zn2+-sensitive N-terminal half, consisting of nesfatin-1 and -2, and the Ca2+-sensitive C-terminal half, consisting of nesfatin-3.

Nucleobindin-2 (Nucb2) is a protein that has been suggested to play roles in a variety of biological processes. Nucb2 contains two Ca2+/Mg2+-binding EF-hand domains separated by an acidic amino acid residue-rich region and a leucine zipper. All of these domains are located within the C-terminal half of the protein. At the N-terminal half, Nucb2 also possesses a putative Zn2+-binding motif. In our recent studies, we observed that Nucb2 underwent Ca2+-dependent compaction and formed a mosaic-like structure consisting of intertwined disordered and ordered regions at its C-terminal half. The aim of this study was to investigate the impact of two other potential ligands: Mg2+, which possesses chemical properties similar to those of Ca2+, and Zn2+, for which a putative binding motif was identified. In this study, we demonstrated that the binding of Mg2+ led to oligomerization state changes with no significant secondary or tertiary structural alterations of Nucb2. In contrast, Zn2+ binding had a more pronounced effect on the structure of Nucb2, leading to the local destabilization of its N-terminal half while also inducing changes within its C-terminal half. These structural rearrangements resulted in the oligomerization and/or aggregation of Nucb2 molecules. Taken together, the results of our previous and current research help to elucidate the structure of the Nucb2, which can be divided into two parts: the Zn2+-sensitive N-terminal half (consisting of nesfatin-1 and -2) and the Ca2+-sensitive C-terminal half (consisting of nesfatin-3). These results may also help to open a new discussion regarding the diverse roles that metal cations play in regulating the structure of Nucb2 and the various physiological functions of this protein.

The Multifaceted Nature of Nucleobindin-2 in Carcinogenesis.

Nucb2 is a multifunctional protein associated with a variety of biological processes. Multiple studies have revealed that Nucb2, and its derivative nesfatin-1, are involved in carcinogenesis. Interestingly, the role of Nucb2/nesfatin-1 in tumorigenesis seems to be dual-both pro-metastatic and anti-metastatic. The implication of Nucb2/nesfatin-1 in carcinogenesis seems to be tissue dependent. Herein, we review the role of Nucb2/nesfatin-1 in both carcinogenesis and the apoptosis process, and we also highlight the multifaceted nature of Nucb2/nesfatin-1.

Molecular mechanism of calcium induced trimerization of C1q-like domain of otolin-1 from human and zebrafish.

The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca2+ and stabilizes its collagen-like triple helix. The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia.

Liquid-liquid phase separation of the intrinsically disordered AB region of hRXRγ is driven by hydrophobic interactions.

Nuclear receptors (NRs) are a family of transcription factors that are regulated endogenously by small lipophilic ligands. Recently, liquid-liquid phase separation (LLPS) has appeared as a new aspect of NR function. In the human retinoid X receptor γ (hRXRγ), the inherently disordered AB region undergoes LLPS via homotypic multivalent interactions. To better understand the functions of liquid condensates, a clear view of the molecular interactions underlying the LLPS are required. The phase separation propensity of the AB region of hRXRγ (AB_hRXG) at a high NaCl concentration, a lower critical solution temperature behavior, and also sensitivity to kosmotropic salts and 1,6-hexanediol, which all indicate the importance of hydrophobic interactions in the formation of AB_hRXG liquid condensates, is presented in the paper. Additionally, molecular crowding agents and TMAO shift the equilibrium, in turn enabling phase transition at lower AB_hRXG concentrations. Although the LLPS of the proteins can lead to aggregation, AB_hRXG liquid condensates are not aggregation prone. Interestingly, the formation of AB_hRXG liquid condensates has an impact on the rest of the receptor, as AB_hRXG liquid condensates recruit the remaining fragment of hRXRγ into the droplets. The ability of AB_hRXG to undergo LLPS might be important for gene expression regulation.

Metal Ions Induce Liquid Condensate Formation by the F Domain of Aedes aegypti Ecdysteroid Receptor. New Perspectives of Nuclear Receptor Studies.

The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the Aedes aegypti mosquito (AaFEcR), the vector of the world's most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The perspectives of this finding on future research on the F domain are discussed, especially in relation to other NR members.