Phosphoserine Residues Research Articles

Novel CDKL5 targets identified in human iPSC-derived neurons

CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.

Aggregation behavior of micellar casein during in vitro simulated digestion triggered by serum Ca2+ in the gastric fluids

The gastric aggregation characteristics of micellar casein (MC) are closely related to the surrounding digestive environment. Together with pepsin and gastric acid, the effect of serum Ca2+ in a digestive fluid is inevitable for the formation of those aggregates. In this study, MC powders with different levels of colloidal calcium phosphate (CCP) were controlled by quick-freezing process. The unique aggregation behaviors accompanied by the addition of exogenous Ca2+ during in vitro simulated digestion were investigated. The results showed that quick-freezing could significantly reduce the CCP level up to 50 % compared to controls. The dissociated micelle structure provided plenty of exposed binding sites, which was prone to recombine with serum Ca2+ in gastric fluids and thus formed the oversized nanoclusters and massive aggregates in stomach. Only about 40 % of the aggregates were dissolved and hydrolyzed after 120 min of gastric digestion. The results of wide-angle coupled small-angle X-ray scattering demonstrated that serum Ca2+ triggered aggregation behaviors largely depend on the pepsin and gastric acids. The para-κ-casein produced by pepsin could be quickly cross-linked by Ca2+-induced connections; while the exposed phosphoserine residues caused by gastric acids allowed more monomers to become nanoclusters by the effect of Ca2+ bridges.

Interfacial network formation by casein blends in the presence of Ca2+ is dominated by β- and κ-casein

With the increasing interest in precision fermentation for food purposes, the opportunity arises to produce individual casein fractions or specific blends based on the requirements of an application. Therefore, the functionality of individual casein fractions has gained attention in academia and industry.This study focused on the air-water interfacial functionality of individual bovine casein fractions in casein blends, to serve as a benchmark for precision fermented casein fractions. With oscillating bubble tensiometry, we showed that the interfacial behavior of a milk-like casein blend of αs1-, αs2-, β-, &κ-casein (ratio 4:1.1:3.8:1.3) was dominated by the β- and κ-casein fractions through the formation of a disordered solid-like viscoelastic interface. The presence of Ca2+ in the buffer was essential; Without Ca2+, the casein blend formed a weak interfacial microstructure where the viscoelastic modulus was dominated by exchange between bulk and interface. The interfacial microstructure was imaged with Langmuir-Blodgett deposition followed by atomic force microscopy. Here we showed that the interfacial structure of β+κ-casein was highly similar to the milk-like casein blend. In the presence of Ca2+ in the buffer, the interfacial microstructure of the casein blend was shown to be more connected and homogeneous compared to a buffer without Ca2+. Thereby highlighting the importance of the phosphoserine residues in β-casein.These results show the opportunity to mimic the interfacial functionality of a milk-like casein blend with only a blend of β+κ-casein but that the presence of phosphoserine residues in β-casein is essential for the formation of a stiff interfacial network.

Dry-heat-induced phosphoserine-specific fragmentation of ovalbumin

When subjected to dry-heating, egg white ovalbumin, a phosphoglycoprotein, undergoes fragmentation and forms soluble aggregates. We investigated the mechanisms of dry-heat-induced fragmentation of ovalbumin. SDS-PAGE analysis showed that ovalbumin fragmented into five polypeptides, and their amount increased over 6 h of dry-heat treatment at 120 °C. The fragments contained fewer or no phosphoserine, compared with that in crude ovalbumin. Liquid chromatography-tandem mass spectrometry analysis of tryptic digests revealed that the fragmentation sites were located on phosphoserine residues, S68 and S344. During fragmentation, the phosphoserine residues underwent conversion into dehydroalanine residues, which were subsequently hydrolyzed. The nitrogen from the dehydroalanine became a newly formed terminal amide group on the N-terminal fragment, while the remaining molecule predominantly formed a new terminal pyruvoyl group. Furthermore, the fragments were incorporated into monomers or soluble aggregates of ovalbumin via covalent and non-covalent bonds. This study demonstrated a novel mechanism for dry-heat-induced fragmentation of phosphoproteins.

Abstract PR02: Factors modulating RAF dimerization downstream of RAS – A mechanistic overview

Abstract The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. The activation of this pathway is tightly regulated with appropriate spatial and temporal signaling cues. The central step in activation of this pathway is the dimerization of RAF kinases. Recent advances in biochemical, enzymatic and structural characterization of various multiprotein complexes in this pathway provide insight into various modes of RAF activity modulation. Kinase RAF and its substrate MEK exist as a pre-formed complex prior to pathway activation. Multiple factors prevent MEK phosphorylation by RAF in this pre-formed complex, including phosphorylation of a Serine residue N-terminal to the kinase domain, which enables 14-3-3 to trap RAF as a monomer. One of our intriguing findings is that cellular ATP exerts a negative regulatory effect on RAF kinase and stabilizes the inactive conformation of RAF monomer. We characterize this negative regulatory effect of ATP of RAF kinase structurally by solving a RAF-ATP complex and enzymology. Upon membrane recruitment of RAF by RAS-GTP, dephosphorylation of the above-mentioned serine residue in RAF occurs by a multiprotein complex composed of phosphatase PP1C, a scaffolding protein SHOC2 and RAS-GTP. We solved the structure of this RAS-SHOC2-PP1C complex and performed a thorough enzymatic analysis of this complex for RAF dephosphorylation. Our results show that RAF specificity is determined by SHOC2 and RAS GTP is responsible for spatial localization both inactive RAF and SHOC2/PP1C to the membrane. This dephosphorylation results in RAF being stabilized as a dimer by 14-3-3, which now binds to phospho-serine residues C-terminal to the kinase domain (S729 in BRAF) in 2 RAF molecules. We solve a structure of the RAF-14-3-3 complex. 14-3-3 induced RAF dimerization increases activity of RAF ~500 fold. Further, oncogenic mutations in RAF and MEK occur basally in tumors, and additional mutations are induced upon pathway inhibitor treatment in the clinic, especially for recent KRAS-G12C treatments. These mutations include RAF mutations that either activate or inactivate the kinase activity of the mutated RAF, but nevertheless result in pathway activation. Our data suggests that these mutations appear to tilt the RAF monomer-dimer equilibrium towards RAF dimer. The molecular mechanism of how the MEK mutations active the pathway in poorly understood. Biochemical and structural characterization of these mutations occurring in MEK kinases suggests that the mechanism of activation is also likely functioning via promoting RAF dimerization. This RAF dimer promotion appears to mechanistically work by relieving the negative regulatory effect of RAF by ATP to result in the “just-right” amount of pathway activation to sustain tumor growth. A comprehensive understanding of the various factors modulating RAF dimerization in the context of normal cell and cancer cells that depend on this pathway can pave the way for more precise interventions for treatment of cancers that depend on the RAS pathway. Citation Format: Jawahar Sudhamsu, Nicholas Liau, Timothy Wendorff, Saeed Izadi, Luca Gerosa. Factors modulating RAF dimerization downstream of RAS – A mechanistic overview [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR02.

Immobilization of silver ions onto casein

In this study, the physicochemical and biological properties of silver-casein complexes were investigated. The casein modification was carried out by a novel method, i.e., at basic conditions (pH 8) using silver precursor in a form of ammonia complex [Ag(NH3)2]+. The selected experimental conditions allowed us to obtain high colloidal stability of protein suspension and also minimized the risk of metal secondary reduction. Subsequently, the mechanism of silver immobilization was evaluated by using a batch sorption study (Freundlich, Langmuir isotherm modeling). Moreover, the influence of accompanying the protein ions on the silver binding process was studied (using ultrafiltration). Results revealed, that casein after the desalting step has much higher silver-binding capacity (258.2 mg/g) compared to untreated protein (157.1 mg/g). The nature of casein-silver interactions was comprehensively studied by multiple instrumental techniques, namely, spectrometric (ICP-MS), spectroscopic (DLS, ATR-FTIR, Fluorescence Spectroscopy), electron microscopy (STEM, SEM-EDX), and separation (SDS-PAGE). Results indicated, that silver sorption had a complex character. The heterogeneous character of metal binding processes was discovered. The DLS studies pointed out the great role of phosphoserine residues in silver-casein interactions and underlined their impact on the micelle size and structure. Moreover, STEM images confirmed the growth of silver nanoparticles upon metal binding to casein. According to FTIR analysis, carboxylic groups of aspartic and glutamic acid were identified as potential metal-binding sites which also could participate in silver reduction. Peptic digestion studies showed a relevant influence of silver incorporation on the structural stability of caseins. Finally, the biological activity of the 600Ag-CN complex was evaluated. Results indicated that the complex effectively inhibited the growth of both Gram-positive (S. aureus) and Gram-negative (K. pneumoniae) bacteria. Still, the treatment of mouse fibroblast cell line L929 with analyzed complex (0.32 mg/mL of 600Ag-CN corresponding to 7.5 µg/mL of silver) decrease cell viability up to 60 %. The toxic activity of the complex may be connected to the Ag+ release. The silver desorption study showed that 600Ag-CN complex released 21.5 %, 49.3 %, and 68.7 % in pH 6.8, 4.5, and 1.2 respectively.

Calcium interactions in amelogenin-derived peptide assembly.

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register β-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.

Interactions between 14-3-3 Proteins and Actin Cytoskeleton and Its Regulation by microRNAs and Long Non-Coding RNAs in Cancer

14-3-3s are a family of structurally similar proteins that bind to phosphoserine or phosphothreonine residues, forming the central signaling hub that coordinates or integrates various cellular functions, thereby controlling many pathways important in cancer, cell motility, cell death, cytoskeletal remodeling, neuro-degenerative disorders and many more. Their targets are present in all cellular compartments, and when they bind to proteins they alter their subcellular localization, stability, and molecular interactions with other proteins. Changes in environmental conditions that result in altered homeostasis trigger the interaction between 14-3-3 and other proteins to retrieve or rescue homeostasis. In circumstances where these regulatory proteins are dysregulated, it leads to pathological conditions. Therefore, deeper understanding is needed on how 14-3-3 proteins bind, and how these proteins are regulated or modified. This will help to detect disease in early stages or design inhibitors to block certain pathways. Recently, more research has been devoted to identifying the role of MicroRNAs, and long non-coding RNAs, which play an important role in regulating gene expression. Although there are many reviews on the role of 14-3-3 proteins in cancer, they do not provide a holistic view of the changes in the cell, which is the focus of this review. The unique feature of the review is that it not only focuses on how the 14-3-3 subunits associate and dissociate with their binding and regulatory proteins, but also includes the role of micro-RNAs and long non-coding RNAs and how they regulate 14-3-3 isoforms. The highlight of the review is that it focuses on the role of 14-3-3, actin, actin binding proteins and Rho GTPases in cancer, and how this complex is important for cell migration and invasion. Finally, the reader is provided with super-resolution high-clarity images of each subunit of the 14-3-3 protein family, further depicting their distribution in HeLa cells to illustrate their interactions in a cancer cell.

Phylogenetic comparative analysis: Chemical and biological features of caseins (alpha-S-1, alpha-S-2, beta- and kappa-) in domestic dairy animals.

Caseins determine the physicochemical, physiological, and biological characteristics of milk. Four caseins—alpha-S-1, alpha-S-2, beta, and kappa—were analyzed phylogenetically and in silico and characterized regarding chemical, antimicrobial, and antioxidant features in five dairy animals: Arabian camels, sheep, goats, cattle, and water buffalos. The sequence of full-length amino acids of the four caseins for the five species was retracted from the NCBI GenBank database. Multiple sequence alignment is used to examine further the candidate sequences for phylogenetic analysis using Clustal X and NJ-Plot tools. The results revealed that sheep and goats possess strong similarities (98.06%) because of their common ancestor. The same was observed with cattle and water buffalos (96.25%). The Arabian camel was located in a single subclade due to low similarity in casein residues and compositions with other dairy animals. Protein modeling showed that alpha-S1- and alpha-S2-caseins possess the highest number of phosphoserine residues. The in silico computed chemical properties showed that β-casein recorded highest hydrophobicity index and lowest basic amino acid content, while α-S2-casein showed the opposite. The computed biological parameters revealed that α-S2-casein presented the highest bactericidal stretches. Only Arabian camel β-casein and k-casein showed one bactericidal stretches. The analysis also revealed that β-casein, particularly in Arabian camels, possesses the highest antioxidant activity index. These results support the importance of the bioinformatics resources to determine milk casein micelles' chemical and biological activities.

Structural basis for SHOC2 modulation of RAS signalling

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers1–3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5–8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11–13, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Atomic force microscopy to assess the mechanical properties of individual casein micelles

Casein micelles (CMs) are ∼100 nm natural colloids found in milk resulting from the complex and unresolved association of casein monomers, phosphorus, and calcium; the latter being either directly bound to the phosphoserine residues of caseins or present as nanoclusters of insoluble micellar calcium phosphate (MCP). Dairy products such as cheese or yogurt are colloidal gels formed by CMs destabilization, for which texture and ability to be processed are essentially determined by rheological properties. Those properties depend on the physico-chemical conditions used during gel formation (e.g., pH, temperature, ionic content). However, questions remain about the origin of this dependence at the microscopic scale. In particular, we still do not have a clear picture of the specific contributions of the rigidity of the elementary bricks (CMs) in comparison with the inter-particles interactions or their spatial distribution. In this work, Atomic Force Microscopy (AFM) is used to evaluate changes in the nanomechanical properties of single CMs following physico-chemical modifications that are known to affect the MCP content and the rheology of the enzymatic milk gel (i.e. decrease of pH and CaCl2 addition). AFM is used as a direct indenter that assesses the CMs’ elastic deformation and gives an estimate of their Young modulus. We show that for a ±18–24% w/w depletion or enrichment in MCP content, the Young modulus of CMs significantly decreases or increases, respectively. This correlation suggests that variations in the modulus of individual CMs could explain the changes in the macroscopic properties of the enzymatic milk gel upon variations of physico-chemical conditions.

Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity.

Anti-tumour inflammatory cytokines are highly toxic when administered systemically. Here, in multiple syngeneic mouse models, we show that the intratumoural injection of recombinantly expressed cytokines bound tightly to the common vaccine adjuvant aluminium hydroxide (alum) (via ligand exchange between hydroxyls on the surface of alum and phosphoserine residues tagged to the cytokine by an alum-binding peptide) leads to weeks-long retention of the cytokines in the tumours, with minimal side effects. Specifically, a single dose of alum-tethered interleukin-12 induced substantial interferon-γ-mediated T-cell and natural-killer-cell activities in murine melanoma tumours, increased tumour antigen accumulation in draining lymph nodes and elicited robust tumour-specific T-cell priming. Moreover, intratumoural injection of alum-anchored cytokines enhanced responses to checkpoint blockade, promoting cures in distinct poorly immunogenic syngeneic tumour models and eliciting control over metastases and distant untreated lesions. Intratumoural treatment with alum-anchored cytokines represents a safer and tumour-agnostic strategy to improving local and systemic anticancer immunity.

Predicting PY motif-mediated protein-protein interactions in the Nedd4 family of ubiquitin ligases.

The Nedd4 family contains several structurally related but functionally distinct HECT-type ubiquitin ligases. The members of the Nedd4 family are known to recognize substrates through their multiple WW domains, which recognize PY motifs (PPxY, LPxY) or phospho-threonine or phospho-serine residues. To better understand protein interactor recognition mechanisms across the Nedd4 family, we report the development and implementation of a python-based tool, PxYFinder, to identify PY motifs in the primary sequences of previously identified interactors of Nedd4 and related ligases. Using PxYFinder, we find that, on average, half of Nedd4 family interactions are likely PY-motif mediated. Further, we find that PPxY motifs are more prevalent than LPxY motifs and are more likely to occur in proline-rich regions and that PPxY regions are more disordered on average relative to LPxY-containing regions. Informed by consensus sequences for PY motifs across the Nedd4 interactome, we rationally designed a focused peptide library and employed a computational screen, revealing sequence- and biomolecular interaction-dependent determinants of WW-domain/PY-motif interactions. Cumulatively, our efforts provide a new bioinformatic tool and expand our understanding of sequence and structural factors that contribute to PY-motif mediated interactor recognition across the Nedd4 family.

Identification of juvenile hormone-induced posttranslational modifications of methoprene tolerant and Krüppel homolog 1 in the yellow fever mosquito, Aedes aegypti

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SignificanceFemale Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy.

Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling proteins site-specifically with a single lanthanide ion remains an ongoing challenge, especially for proteins that are not suitable for ligation with cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine in conjunction with other negatively charged residues, such as aspartate, glutamate or a second phosphoserine residue. The close proximity of the binding sites to the protein backbone leads to good immobilization of the lanthanide ion, as evidenced by the excellent quality of fits between experimental PCSs and PCSs calculated with a single magnetic susceptibility anisotropy ( tensor. An improved two-plasmid system was designed to enhance the yields of proteins with genetically encoded phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt bridges, although the presence of too many negatively charged residues in close proximity can also lead to unfolding of the protein. In view of the quality of the tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine, this method presents an attractive tool for generating PCSs in stable proteins, particularly as it is independent of cysteine residues.

Study on β-Casein Depleted Casein Micelles: Micellar Stability, Enzymatic Cross-Linking, and Suitability as Nanocarriers.

β-Casein is an amphiphilic protein and thus considered as multilaterally bound in casein micelles. Its polar molecule part, in particular the phosphoserine residues, can interact electrostatically with colloidal calcium phosphate (CCP) to form nanoclusters and its nonpolar molecule part enhances micellar stability by forming hydrophobic bonds to other caseins. Because cooling weakens hydrophobic interactions, a substantial portion of β-casein can be irreversibly removed from the casein micelle by repeated depletion steps, including cooling and subsequent ultracentrifugation. Although this effect of cooling on the micellar β-casein concentration has been well known for decades, the influence of depletion on the main characteristics of casein micelles has been less investigated yet. Therefore, we aimed to analyze the consequences of β-casein depletion on the stability as well as the functionality of casein micelles to evaluate the suitability of depleted compared to native casein micelles as nanocarriers. Up to 43.2% of the total β-casein was irreversibly sequestered from native casein micelles by repeated cooling and ultracentrifugation steps. Depletion showed no effect on size distribution as well as polydispersity and particle concentration of micelle suspensions as measured via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), respectively. Furthermore, the stability of the micelles against ethanol or the chelating agent ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was not influenced by β-casein depletion. Notwithstanding, depleted micelles were less susceptible to enzymatic cross-linking by microbial transglutaminase (mTG), indicating narrowed water channels due to depletion. Additionally, loading experiments showed that depleted micelles could be loaded with linoleic acid (LA) as intensively as native micelles, whereupon LA displaces up to 81.3% of β-casein from native micelles. Our results confirm that depletion does not enhance the ability of the casein micelle to act as a nanocarrier for hydrophobic substances but could support the understanding of the casein micelle structure. Based on the observed unchanged stability against EGTA, the hindered enzymatical cross-linking, and the efficient displacing of β-casein by LA, we suggest that the major portion of micellar β-casein is hydrophobically incorporated into the micelle structure without impact on the formation of calcium phosphate nanoclusters. The main role of β-casein for the casein micelle structure, therefore, might be to facilitate the high hydration of the interior and thus the high permeability of casein micelles.

Addition of calcium and magnesium chlorides as simple means of varying bound and precipitated minerals in casein micelle: Effect on enzymatic coagulation

In casein micelle (CM), Ca is either precipitated in the colloidal calcium phosphate (CCP) stabilized by clusters of phosphoserine (SEP) residues, or is directly bound to SEP (or glutamic and aspartic acids) of caseins without inorganic phosphate. However, it is currently not possible to titrate separately the different micellar Ca forms, making it difficult to assess their respective importance for CM properties and behavior. Both Ca2+ and Mg2+ have the same binding constants with SEP. Moreover, MgHPO4 is more soluble than CaHPO4, and its natural concentration in milk is lower. Thus, upon addition of MgCl2, Mg is mainly exchanged with CM in the bound form, whereas upon addition of CaCl2, Ca is mainly exchanged in the precipitated form. Our objective was to assess the role of the 2 forms of micellar cations (bound and precipitated) during the enzymatic coagulation of cow milk. Magnesium chloride, CaCl2, or KCl (10 mM) were added to milk and pH was adjusted to 6.6 after overnight equilibration. The KCl-supplemented milk was a positive control to assess the effect of the increased ionic strength after MgCl2 and CaCl2 addition. Mineral partition between soluble and colloidal phases after salt addition was assessed both experimentally and by using computer simulation. Enzymatic coagulation was proceeded at 30°C. Hydrolysis of κ-casein was followed by the quantitative determination of caseinomacropeptide released by RP-HPLC, aggregation of para-κ-casein micelles was measured through the evolution of backscattering properties of milk and the gel time and gel firming kinetics were determined using a Chymograph (Chr. Hansen, Horsholm, Denmark). The KCl addition did not affect mineral partition. As expected, CaCl2 addition mainly increased the CCP content, whereas the addition of MgCl2 mainly increased the bound divalent cations content. The kinetics of κ-casein hydrolysis was slowed down after CaCl2 and MgCl2 addition, and was negatively correlated with the concentration of divalent cations in the soluble phase of milk. Aggregation and gel firming curves plotted versus the progress of κ-casein hydrolysis were similar for both CaCl2- and MgCl2-supplemented milk. In view of the dual-binding model for CM assembly, this means that both Ca forms reduce electronegative repulsions between para-micelles by specific charge shielding. Inclusion of 2 Ca forms in structural models for CM allows a more detailed comprehension of how mineral equilibria affect CM properties.

Peptides from diatoms and grasses harness phosphate ion binding to silica to help regulate biomaterial structure

Many life forms generate intricate submicron biosilica structures with various important biological functions. The formation of such structures, from the silicic acid in the waters and in the soil, is thought to be regulated by unique proteins with high repeats of specific amino acids and unusual sidechain modifications.Some silicifying proteins are characterized by high prevalence of basic amino acids in their primary structures. Lysine-rich domains are found, for instance, in diatom silaffin proteins and in the sorghum grass siliplant1 protein. These domains exhibit catalytic activity in silica chain condensation, owing to molecular interactions of the lysine amine groups with the forming mineral. The use of amine chemistry by two very remote organisms has motivated us to seek other molecular biosilicification processes that may be common to the two life forms.In diatom silaffins, domains rich in phosphoserine residues are thought to assist the assembly of silaffin molecules into an organic supra-structure which serves as a template for the silica to precipitate on. This mold, held by salt bridges between serine phosphates and lysine amines, dictates the shape of the silica particles formed. Yet, silica synthesized with the dephosphorylated silaffin in phosphate buffer showed similar morphology to the one prepared with the native protein, suggesting that a defined spatial arrangement of serine phosphates is not required to generate silica with the desired shape. Concurrently, free phosphates enhanced the activity of siliplant1 in silica formation.It is therefore beneficial to characterize the involvement of these anions as co-factors in regulated silicification by functional peptides from the two proteins and to understand whether they play similar molecular role in the mechanism of mineralization.Here we analyze the molecular interactions of free phosphate ions with silica and the silaffin peptide PL12 and separately with silica and siliplant1 peptide SLP1 in the two biomimetic silica products generated by the two peptides. MAS NMR measurements show that the phosphate ions interact with the peptides and at the same time may be forming bonds with the silica mineral. This bridging capability may add another avenue by which the structure of the silica material is influenced. A model for the molecular/ionic interactions at the bio-inorganic interface is described, which may have bearings for the role of phosphorylated residues beyond the function as intermolecular cross linkers or free phosphate ions as co-factors in regulation of silicification. Statement of SignificanceThe manuscript addresses the question how proteins in diatoms and plants regulate the biosilica materials that are produced for various purposes in organisms. It uses preparation of silica in vitro with functional peptide derivatives from a sorghum grass protein and from a diatom silaffin protein separately to show that phosphate ions are important for the control that is achieved by these proteins on the final shape of the silica material produced. It portrays via magnetic resonance spectroscopic measurements, in atomic detail, the interface between atoms in the peptide, atoms on the surface of the silica formed and the phosphate ions that form chemical bonds with atoms on the silica as part of the mechanism of action of these peptides.

A Chemical Probe for Dehydrobutyrine.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.