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Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins – the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules in vitro assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in E. coli after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.

Ameloblastin (Ambn) is an intrinsically disordered protein (IDP) with a specific function of forming heterogenous homooligomers. The oligomeric function is led through a specific sequence encoded by exon 5 of Ambn. Due to the IDP character of Ambn to form oligomers, protein purification is subject to many challenges. Human ameloblastin (AMBN) and its two isoforms, I and II have already been purified as a recombinant protein in a bacterial expression system and functionally characterized in vitro. However, here we present a new purification protocol for the production of native AMBN in its original formation as a homooligomeric heterogeneous IDP. The purification process consists of three chromatographic steps utilizing His-tag and Twin Strep-tag affinity chromatography, along with size exclusion and reverse affinity chromatography. The presented workflow offers the production of AMBN in sufficient yield for in vitro protein characterizations and can be used to produce both AMBN isoforms I and II. • The production of ameloblastin (AMBN) protein in its native heterogenous homo-oligomeric state is subject to many challenges. • We report the first AMBN native purification in a three-step protocol consisting of chromatographic steps along with SEC and reverse IMAC. • The final yield of AMBN is sufficient for in vitro protein characterizations and can be used to produce both AMBN isoforms I and II. [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin (TRPM) channels, a subfamily of the TRP superfamily, constitute a diverse group of ion channels involved in mediating crucial cellular processes like calcium homeostasis. These channels exhibit complex regulation, and one of the key regulatory mechanisms involves their interaction with calmodulin (CaM), a cytosol ubiquitous calcium-binding protein. The association between TRPM channels and CaM relies on the presence of specific CaM-binding domains in the channel structure. Upon CaM binding, the channel undergoes direct and/or allosteric structural changes and triggers down- or up-stream signaling pathways. According to current knowledge, ion channel members TRPM2, TRPM3, TRPM4, and TRPM6 are directly modulated by CaM, resulting in their activation or inhibition. This review specifically focuses on the interplay between TRPM channels and CaM and summarizes the current known effects of CaM interactions and modulations on TRPM channels in cellular physiology.

Melastatin transient receptor potential (TRPM) channels belong to one of the most significant subgroups of the transient receptor potential (TRP) channel family. Here, we studied the TRPM5 member, the receptor exposed to calcium-mediated activation, resulting in taste transduction. It is known that most TRP channels are highly modulated through interactions with extracellular and intracellular agents. The binding sites for these ligands are usually located at the intracellular N- and C-termini of the TRP channels, and they can demonstrate the character of an intrinsically disordered protein (IDP), which allows such a region to bind various types of molecules. We explored the N-termini of TRPM5 and found the intracellular regions for calcium-binding proteins (CBPs) the calmodulin (CaM) and calcium-binding protein S1 (S100A1) by in vitro binding assays. Furthermore, molecular docking and molecular dynamics simulations (MDs) of the discovered complexes confirmed their known common binding interface patterns and the uniqueness of the basic residues present in the TRPM binding regions for CaM/S100A1.

Transient receptor potential melastatin 7 (TRPM7) represents melastatin TRP channel with two significant functions, cation permeability and kinase activity. TRPM7 is widely expressed among tissues and is therefore involved in a variety of cellular functions representing mainly Mg 2+ homeostasis, cellular Ca 2+ flickering, and the regulation of DNA transcription by a cleaved kinase domain translocated to the nucleus. TRPM7 participates in several important biological processes in the nervous and cardiovascular systems. Together with the necessary function of the TRPM7 in these tissues and its recently analyzed overall structure, this channel requires further studies leading to the development of potential therapeutic targets. Here we present the first study investigating the N-termini of TRPM7 with binding regions for important intracellular modulators calmodulin (CaM) and calcium-binding protein S1 (S100A1) using in vitro and in silico approaches. Molecular simulations of the discovered complexes reveal their potential binding interfaces with common interaction patterns and the important role of basic residues present in the N-terminal binding region of TRPM., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi-domain protein system. The simple model system of the two-domain fusion protein investigated in this work consisted of a well-folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3-TrpCage, TrpCage-PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics., (© 2021 The Protein Society.)

Constantly increasing attention to bioengineered proteins has led to the rapid development of new functional targets. Here we present the biophysical and functional characteristics of the newly designed CaM/AMBN-Ct fusion protein. The two-domain artificial target consists of calmodulin (CaM) and ameloblastin C-terminus (AMBN-Ct). CaM as a well-characterized calcium ions (Ca 2+ ) binding protein offers plenty of options in terms of Ca 2+ detection in biomedicine and biotechnologies. Highly negatively charged AMBN-Ct belongs to intrinsically disordered proteins (IDPs). CaM/AMBN-Ct was designed to open new ways of communication synergies between the domains with potential functional improvement. The character and function of CaM/AMBN-Ct were explored by biophysical and molecular modelling methods. Experimental studies have revealed increased stability and preserved CaM/AMBN-Ct function. The results of molecular dynamic simulations (MDs) outlined different interface patterns between the domains with potential allosteric communication within the fusion., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca 2+ )-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca 2+ may explain the role of AMBN in biomineralization and more generally in Ca 2+ homeostasis processes.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP 2 ). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP 2 . The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.