A Conserved Ribosomal Protein Has Entirely Dissimilar Structures in Different Organisms.

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-termini and C-termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis.

Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling.

Intracellular microbial rhodopsin-based optogenetics to control metabolism and cell signaling.

Microbial rhodopsin (MRs) ion channels and pumps have become invaluable optogenetic tools for neuroscience as well as biomedical applications. Recently, MR-optogenetics expanded towards subcellular organelles opening principally new opportunities in optogenetic control of intracellular metabolism and signaling via precise manipulations of organelle ion gradients using light. This new optogenetic field expands the opportunities for basic and medical studies of cancer, cardiovascular, and metabolic disorders, providing more detailed and accurate control of cell physiology. This review summarizes recent advances in studies of the cellular metabolic processes and signaling mediated by optogenetic tools targeting mitochondria, endoplasmic reticulum (ER), lysosomes, and synaptic vesicles. Finally, we discuss perspectives of such an optogenetic approach in both fundamental and applied research.

Intrinsic factors behind long COVID: III. Persistence of SARS-CoV-2 and its components.

Considerable research has been done in investigating SARS-CoV-2 infection, its characteristics, and host immune response. However, debate is still ongoing over the emergence of post-acute sequelae of SARS-CoV-2 infection (PASC). A multitude of long-lasting symptoms have been reported several weeks after the primary acute SARS-CoV-2 infection that resemble several other viral infections. Thousands of research articles have described various post-COVID-19 conditions. Yet, the evidence around these ongoing health problems, the reasons behind them, and their molecular underpinnings are scarce. These persistent symptoms are also known as long COVID-19. The persistence of SARS-CoV-2 and/or its components in host tissues can lead to long COVID. For example, the presence of viral nucleocapsid protein and RNA was detected in the skin, appendix, and breast tissues of some long COVID patients. The persistence of viral RNA was reported in multiple anatomic sites, including non-respiratory tissues such as the adrenal gland, ocular tissue, small intestine, lymph nodes, myocardium, and sciatic nerve. Distinctive viral spike sequence variants were also found in non-respiratory tissues. Interestingly, prolonged detection of viral subgenomic RNA was observed across all tissues, sometimes in multiple tissues of the same patient, which likely reflects recent but defective viral replication. Moreover, the persistence of SARS-CoV-2 RNA was noticed throughout the brain at autopsy, as late as 230 days following symptom onset among unvaccinated patients who died of severe infection. Here, we review the persistence of SARS-CoV-2 and its components as an intrinsic factor behind long COVID. We also highlight the immunological consequences of this viral persistence.

Intrinsic factors behind long COVID: IV. Hypothetical roles of the SARS-CoV-2 nucleocapsid protein and its liquid-liquid phase separation.

When the SARS-CoV-2 virus infects humans, it leads to a condition called COVID-19 that has a wide spectrum of clinical manifestations, from no symptoms to acute respiratory distress syndrome. The virus initiates damage by attaching to the ACE-2 protein on the surface of endothelial cells that line the blood vessels and using these cells as hosts for replication. Reactive oxygen species levels are increased during viral replication, which leads to oxidative stress. About three-fifths (~60%) of the people who get infected with the virus eradicate it from their body after 28 days and recover their normal activity. However, a large fraction (~40%) of the people who are infected with the virus suffer from various symptoms (anosmia and/or ageusia, fatigue, cough, myalgia, cognitive impairment, insomnia, dyspnea, and tachycardia) beyond 12 weeks and are diagnosed with a syndrome called long COVID. Long-term clinical studies in a group of people who contracted SARS-CoV-2 have been contrasted with a noninfected matched group of people. A subset of infected people can be distinguished by a set of cytokine markers to have persistent, low-grade inflammation and often self-report two or more bothersome symptoms. No medication can alleviate their symptoms efficiently. Coronavirus nucleocapsid proteins have been investigated extensively as potential drug targets due to their key roles in virus replication, among which is their ability to bind their respective genomic RNAs for incorporation into emerging virions. This review highlights basic studies of the nucleocapsid protein and its ability to undergo liquid-liquid phase separation. We hypothesize that this ability of the nucleocapsid protein for phase separation may contribute to long COVID. This hypothesis unlocks new investigation angles and could potentially open novel avenues for a better understanding of long COVID and treating this condition.

Amyloid-α Peptide Formed through Alternative Processing of the Amyloid Precursor Protein Attenuates Alzheimer's Amyloid-ß Toxicity via Cross-Chaperoning.

Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-ß (Aß), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aß, aggregates rapidly to form amyloids and can expedite the aggregation of Aß through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aß to form amyloid aggregates that are less toxic than pure Aß aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aß in solution. This is the first evidence that Aα can coassemble with Aß and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.

Reexamining the diverse functions of arginine in biochemistry.

Arginine in a free-state and as part of peptides and proteins shows distinct tendency to form clusters. In free-form, it has been found useful in cryoprotection, as a drug excipient for both solid and liquid formulations, as an aggregation suppressor, and an eluent in protein chromatography. In many cases, the mechanisms by which arginine acts in all these applications is either debatable or at least continues to attract interest. It is quite possible that arginine clusters may be involved in many such applications. Furthermore, it is possible that such clusters are likely to behave as intrinsically disordered polypeptides. These considerations may help in understanding the roles of arginine in diverse applications and may even lead to better strategies for using arginine in different situations.

Solvent polarity and hydrophobicity of solutes are two sides of the same coin.

The hydrophobicity of solutes measures the intensity of a solute's interaction with aqueous environment. The aqueous environment may change with its composition, leading to changes in its solvent properties largely characterized by polarity. As a result, the relative hydrophobicity of a solute is a function of the solute structure and the properties of the water-based solvent determined by the total composition of the aqueous phase. This aspect is commonly ignored by medicinal chemists even though it is essential for drug distribution between different biological tissues. Partitioning of solutes in aqueous two-phase systems provides the relative hydrophobicity estimates for any water-soluble compounds that can be used to improve predictions of the toxicity and other biological effects of these compounds.

Stress-granules, P-bodies, and cell aging: A bioinformatics study.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Small-angle X-ray scattering structural insights into alternative pathway of actin oligomerization associated with inactivated state.

In addition to the well-known monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can exist in the so-called inactivated form (I-actin). Hsp70 chaperon, prefoldin, and CCT chaperonin are required to obtain native globular state. In contrast, I-actin is spontaneously formed in the absence of intracellular folding machinery. I-actin can be obtained from G-actin by elimination of divalent ion, incubation in presence of small concentrations of denaturants, and by heat exposure. Since G-actin is a quasi-stationary, thermodynamically unstable form, it can gradually transform into inactivated state in the absence of chelating/denaturating agents or heat exposure, but the transition is much slower. I-actin was shown to associate into oligomers up to the molecular weight of 14-16 G-actin monomers, though the structure of these oligomers remains uncharacterized. This study employs small-angle X-ray scattering to reveal novel insights into the oligomerization process of such spontaneously formed inactivated actin. These oligomers are differentiated from F-actin through comparative analysis, highlighting a unique oligomerization pathway.

Design of stable circular permutants of the GroEL chaperone apical domain.

Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating protein stability while keeping intra-protein interactions intact. Amidst the creation of circular permutants, determining the optimal placement of the new N- and C-termini stands as a pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT's predictions of disorder propensity to guide the design of circular permutants for the GroEL apical domain (residues 191-345). Our underlying hypothesis posited that a higher predicted disorder value would correspond to reduced stability in the circular permutants, owing to the increased likelihood of fluctuations in the novel N- and C-termini. To substantiate this hypothesis, we engineered six circular permutants, positioning glycines within the loops as locations for the new N- and C-termini. We demonstrated the validity of our hypothesis along the set of the designed circular permutants, as supported by measurements of melting temperatures by circular dichroism and differential scanning microcalorimetry. Consequently, we propose a novel computational methodology that rationalizes the design of circular permutants with projected stability. Video Abstract.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Moonlighting enzymes: when cellular context defines specificity.

It is not often realized that the absolute protein specificity is an exception rather than a rule. Two major kinds of protein multi-specificities are promiscuity and moonlighting. This review discusses the idea of enzyme specificity and then focusses on moonlighting. Some important examples of protein moonlighting, such as crystallins, ceruloplasmin, metallothioniens, macrophage migration inhibitory factor, and enzymes of carbohydrate metabolism are discussed. How protein plasticity and intrinsic disorder enable the removing the distinction between enzymes and other biologically active proteins are outlined. Finally, information on important roles of moonlighting in human diseases is updated.

Chaotic aging: intrinsically disordered proteins in aging-related processes.

The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging. The dataset of 1702 aging-related proteins was collected from established aging databases and experimental studies. There is a noticeable presence of IDPs/IDRs, accounting for about 36% of the aging-related dataset, which is however less than the disorder content of the whole human proteome (about 40%). A Gene Ontology analysis of the used here aging proteome reveals an abundance of IDPs/IDRs in one-third of aging-associated processes, especially in genome regulation. Signaling pathways associated with aging also contain IDPs/IDRs on different hierarchical levels, revealing the importance of "structure-function continuum" in aging. Protein-protein interaction network analysis showed that IDPs present in different clusters associated with different aging hallmarks. Protein cluster with IDPs enrichment has simultaneously high liquid-liquid phase separation (LLPS) probability, "nuclear" localization and DNA-associated functions, related to aging hallmarks: genomic instability, telomere attrition, epigenetic alterations, and stem cells exhaustion. Intrinsic disorder, LLPS, and aggregation propensity should be considered as features that could be markers of pathogenic proteins. Overall, our analyses indicate that IDPs/IDRs play significant roles in aging-associated processes, particularly in the regulation of DNA functioning. IDP aggregation, which can lead to loss of function and toxicity, could be critically harmful to the cell. A structure-based analysis of aging and the identification of proteins that are particularly susceptible to disturbances can enhance our understanding of the molecular mechanisms of aging and open up new avenues for slowing it down.

Formation of Amyloid-Like Conformational States of ß-Structured Membrane Proteins on the Example of OMPF Porin from the Yersinia pseudotuberculosis Outer Membrane.

The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with ß-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total ß-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to ß-barrel.

What Can Be Learned from the Partitioning Behavior of Proteins in Aqueous Two-Phase Systems?

This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.

The Proteomic Analysis of Cancer-Related Alterations in the Human Unfoldome.

Many proteins lack stable 3D structures. These intrinsically disordered proteins (IDPs) or hybrid proteins containing ordered domains with intrinsically disordered protein regions (IDPRs) often carry out regulatory functions related to molecular recognition and signal transduction. IDPs/IDPRs constitute a substantial portion of the human proteome and are termed "the unfoldome". Herein, we probe the human breast cancer unfoldome and investigate relations between IDPs and key disease genes and pathways. We utilized bottom-up proteomics, MudPIT (Multidimensional Protein Identification Technology), to profile differentially expressed IDPs in human normal (MCF-10A) and breast cancer (BT-549) cell lines. Overall, we identified 2271 protein groups in the unfoldome of normal and cancer proteomes, with 148 IDPs found to be significantly differentially expressed in cancer cells. Further analysis produced annotations of 140 IDPs, which were then classified to GO (Gene Ontology) categories and pathways. In total, 65% (91 of 140) IDPs were related to various diseases, and 20% (28 of 140) mapped to cancer terms. A substantial portion of the differentially expressed IDPs contained disordered regions, confirmed by in silico characterization. Overall, our analyses suggest high levels of interactivity in the human cancer unfoldome and a prevalence of moderately and highly disordered proteins in the network.

Stochasticity of Biological Soft Matter: Emerging Concepts in Intrinsically Disordered Proteins and Biological Phase Separation.

At the turn of this century, cardinal changes took place in the perceptions of the structure and function of proteins, as well as in the organizational principles of membrane-less organelles. As a result, the model of the organization of living matter is changing to one described by highly dynamic biological soft matter positioned at the edge of chaos. Intrinsically disordered proteins (IDPs) and membrane-less organelles are key examples of this new outlook and may represent a critical foundation of life, defining its complexity and the evolution of living things.

Mobility and disorder in antibody and antigen binding sites do not prevent immunochemical recognition.

The known polyspecificity of antibodies, which is crucial for efficient immune response, is determined by the conformational flexibility and intrinsic disorder encoded in local peculiarities of the amino acid sequence of antibodies within or in the vicinity of their complementarity determining regions. Similarly, epitopes represent fuzzy binding sites, which are also characterized by local structural flexibility. Existing data suggest that the efficient interactions between antigens and antibodies rely on the conformational mobility and some disorder of their binding sites and therefore can be relatively well described by the "flexible lock - adjustable key" model, whereas both, extreme order (rigid lock-and-key) and extreme disorder (viral shape-shifters) are not compatible with the efficient antigen-antibody interactions and are not present in immune interactions.