Deciphering the dual nature of nesfatin-1: a tale of zinc ion's Janus-faced influence.

BACKGROUND: Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2). RESULTS: Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule. CONCLUSIONS: In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2.

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.

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.

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.

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.

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.

Functional derivatives of human dentin matrix protein 1 modulate morphology of calcium carbonate crystals.

Dentin matrix protein 1 (DMP1) is an acidic, extracellular matrix protein essential for biomineralization of calcium phosphate, in bone and dentin. It is proteolytically processed into two fragments, 44K and 56K. Recently, the presence of DMP1 was noticed in inner ear, specifically in otoconia, which are calcium carbonate biominerals involved in sensing of balance. In this study, the solution structure and biomineralization activity of otoconial 44K and 56K fragments toward calcium carbonate were investigated. The results of analytical ultracentrifugation, circular dichroism, and gel filtration indicated that DMP1 fragments are disordered in solution. Notably, 56K formed oligomers in the presence of calcium ions. It was also observed that both fragments influenced the crystal growth by in vitro biomineralization assay and scanning electron microscopy. In addition, they sequester the calcium ions during the calcite formation. Calcium carbonate crystals precipitated in vitro changed their size and shape in the presence of DMP1 fragments. Oligomerization propensity of 56K may significantly enhance this function. Our study indicates that intrinsically disordered DMP1 has a previously unknown regulatory function for biomineralization of otoconia.

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.

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.

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).

In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ozyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

The intrinsically disordered region of GCE protein adopts a more fixed structure by interacting with the LBD of the nuclear receptor FTZ-F1.

The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.

Membraneless organelles and liquid-liquid phase separation ­ methods for their characterisation / Organella bezblonowe a separacja faz ciecz-ciecz ­ metody ich badan.

Membraneless organelles (MLOs) are a large group of intracellular compartments formed during various stages of a cell life. They are important subcellular structures which enable a cell performance of vital physiological processes including stress response. MLOs can be found in cytoplasm and organelles that are sealed by lipidic membrane, mainly in nucleus. They are formed by the thermodynamically driven liquid-liquid phase separation (LLPS). MLOs contain proteins possessing intrinsically disordered regions (IDRs) which together with RNA spontaneously phase separate from the surrounding milieu. This paper presents information on the biophysical basses of the formation and functionality of MLOs. It also discusses a range of experimental techniques that can be applied in biochemical and biological studies of these sub-cellular structures.

Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain-containing protein 4 (NPAS4).

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic ß-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.

Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm-l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm-l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein-enriched region flanked by protein-depleted regions. It can be explained as a result of the Ostwald-like ripening process and intrinsic properties of Stm-l, and bears some analogy to the daily growth layers of the otolith.

Copper(II)-Binding Induces a Unique Polyproline Type II Helical Structure within the Ion-Binding Segment in the Intrinsically Disordered F-Domain of Ecdysteroid Receptor from Aedes aegypti.

Reproduction of the dominant vector of Zika and dengue diseases, Aedes aegypti mosquito, is controlled by an active heterodimer complex composed of the 20-hydroxyecdysone receptor (EcR) and ultraspiracle protein. Although A. aegypti EcR shares the structural and functional organization with other nuclear receptors, its C-terminus has an additional long F domain (AaFEcR). Recently, we showed that the full length AaFEcR is intrinsically disordered with the ability to specifically bind divalent metal ions. Here, we describe the details of the exhaustive structural and thermodynamic properties of Zn2+- and Cu2+-complexes with the AaFEcR domain, based on peptide models of its two putative metal binding sites (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2). Unexpectedly, only in the presence of increasing concentrations of Cu2+ ions, the Ac-HGPHPHPHG-NH2 peptide gained a metal ion-induced poly-l-proline type II helical structure, which is unique for members of the family of nuclear receptors.

Molecular determinants of Drosophila immunophilin FKBP39 nuclear localization.

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 µM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 µM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti.

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD-NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.