How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a "grappling hook" mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT * . The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT * domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT * . In summary, we demonstrate how inducing co-translational folding via a molecular “spindle and thread” mechanism can overcome poor translation efficiency of partially disordered proteins.

Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid‐like aggregation during spinning. Here, we elucidate the stability of the highly soluble N‐terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates. Surprisingly, we find that NT, despite being largely composed of amyloidogenic sequences, showed no signs of concentration‐dependent aggregation. Using a novel intracellular hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approach, we reveal that NT adopts a tight fold in the E. coli cytosol and in this manner conceals its aggregation‐prone regions by maintaining a tight fold under crowded conditions. Fusion of NT to the unstructured amyloid‐forming Aβ40 peptide, on the other hand, results in the formation of fibrillar aggregates. However, HDX‐MS indicates that the NT domain is only partially incorporated into these aggregates in vivo. We conclude that NT is able to control its aggregation to remain functional under the extreme conditions in the spider silk gland.

R-phycoerythrin is a fluorescent red protein-pigment complex present in red algae. It belongs to the light-harvesting phycobiliprotein family with phycocyanin and allophycocyanin, which together make up the phycobilisome. The protein is most commonly used for its fluorescence properties in biochemistry. To determine the highest R-phycoerythrin yielding red algal species, a reliable quantification method is required. Calculating R-phycoerythrin yield from the crude extract’s absorption values has been the quickest and most widely used technique but at the same time, often misleading approach when impurities are present. Small phycoerythrin, which is often present in red algal suspensions, alters the absorption curve and leads to erroneous results. In the given study, we utilized an HPLC-SEC method with fluorescence and photo-diode array detectors, which is capable of separating the phycobiliproteins from interfering compounds. Also, the technique can detect and quantitate R-phycoerythrin yields reliably. A total of nine red algal species from Estonian and Japanese waters were tested, and small phycoerythrin was detected from most of the samples. For two of the tested species, the spectrophotometric quantification methods overestimated the yields by more than 200%.

Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high β-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer’s disease associated amyloid-β peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces Aβ42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.

Phycobiliproteins, red phycoerythrins and blue (allo)phycocyanins, are one of the most highly valued components extracted from cyanobacteria and red algae. In the given study, 95 Furcellaria lumbricalis and 92 Coccotylus truncatus samples were collected from different locations and depths from Kassari Bay, Estonia. Firstly, a strong, positive exponential correlation between the R-phycoerythrin and allophycocyanin concentrations and the collection depth (R > 0.75 and R > 0.61, respectively) was found. Secondly, a preparative scale purification was performed, and the proteins were further characterised by spectrophotometry, fluorimetry and native mass spectrometry. The obtained purity indices (A(566)/A(280)) for the R-phycoerythrins were 1.6 for F. lumbricalis and 2.6 for C. truncatus, thus making them suitable for cosmetics and food industries. Purified R-phycoerythrins from the two algal species had subtle differences in absorption and fluorescence maxima. Based on their slightly different absorption characteristics, R-phycoerythrin from F. lumbricalis would categorise as type III (RIII-PE) and R-phycoerythrin from C. truncatus, alongside with the Sigma 52412 standard, as type II (RII-PE). The native mass spectrometry analysis supported the results by showing different intact complexes with distinct gamma-subunits for the R-phycoerythrin types. The R-phycoerythrin hexamers linked to various gamma-subunits, which are in turn coupled to different chromophores, could explain the similarities (analytical standard and C. truncatus) and differences (F. lumbricalis) in absorption and fluorescence characteristics among the tested species.

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine- N -oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation. ᅟ

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

High level of positive affectivity acts as a protective factor against adverse effects of stress and decreases vulnerability to mood disorders and drug abuse. Fifty-kHz ultrasonic vocalizations (50-kHz USV) index the level of positive affect in the rat, whereas stable, trait-like inter-individual differences in terms of vocalization activity exist. Previously we have demonstrated that chronic stress can alter the effect of repeated amphetamine administration on 50-kHz vocalizations, and this effect is different in rats with high and low positive affectivity. In the present study it was tested whether the chronic stress effect on amphetamine-induced 50-kHz USV activity is altered by inhibition of serotonin reuptake. Male Wistar high (HC) and low (LC) 50-kHz vocalizing rats were subjected to 43-day chronic variable stress (CVS) regimen. On day 17 of the CVS, the four-week once a day fluoxetine (10 mg/kg) treatment was started. After the CVS and fluoxetine treatment, amphetamine (1 mg/kg) was daily administered for ten days and again nine days after withdrawal. Chronically stressed rats developed cross-sensitization of 50-kHz USV-s with repeated administration of amphetamine except the stressed LC rats that had not received fluoxetine. Amphetamine treatment decreased serotonin turnover in the fluoxetine-treated HC rats, but increased it in fluoxetine-treated LC rats. The effect of amphetamine on levels of amino acids in frontal cortex and hippocampus also depended on previous experience with chronic stress, repeated treatment with fluoxetine, and positive affectivity. Hence, this study provides further evidence the effects of chronic stress, psychostimulants, and a selective serotonin reuptake inhibitor are influenced by the inherent positive affectivity.

Phycobiliproteins are natural additives in food industry and essential fluorescent probes in biotechnology. Various algal species may be suitable for their extraction, therefore, it is important to study different seaweeds to determine the highest-yielding species. Furcellaria lumbricalis, a red algal species primarily harvested from Canadian, Danish and Estonian waters, has been utilized for merely furcellaran extraction in Estonia on a commercial scale for over 50 years. To further valorize the bioresource it is vital to study whether additional products, such as phycobiliproteins, can be extracted. In the given study, optimal phycobiliprotein extraction conditions from F. lumbricalis were determined for the first time. For quantification, a novel high-pressure liquid chromatography method with fluorescence and photodiode array detection was developed. Considering R-phycoerythrin, citrate pH 6 buffer gave the highest yields (0.13% using fluorescence detector and 0.43% using photodiode array detector) after an extraction for 24 h at 20 degrees C. Different enzymes (cellulases, xylanases, galactosidases) and their combinations, lowering the extraction temperature or ultrasonication increased the yield further. In addition, red-coloured low-molecular fraction (with similar absorption characteristics to R-phycoerythrin) was separated. For allophycocyanin phosphate pH 6 buffer gave slightly higher yield (0.09% using fluorescence detector and 0.12% using photodiode array detector), compared to citrate or acetate buffer at the same pH. However, the tested enzyme combination, temperature variation and ultrasonication were not so advantageous for allophyocyanin recovery. The phycocyanin concentration from the red algae was below the limit of detection.

Alzheimer’s disease (AD) is the most common form of dementia and there is no successful treatment available. Evidence suggests that fibril formation of the amyloid β-peptide (Aβ) is a major underlying cause of AD, and treatment strategies that reduce the toxic effects of Aβ amyloid are sought for. The BRICHOS domain is found in several proteins, including Bri2 (also called integral membrane protein 2B (ITM2B)), mutants of which are associated with amyloid and neurodegeneration, and Bri3 (ITM2C). We have used mouse hippocampal neurons and brain tissues from mice and humans and show Bri3 deposits dispersed on AD plaques. In contrast to what has been shown for Bri2, Bri3 immunoreactivity is decreased in AD brain homogenates compared to controls. Both Bri2 and Bri3 BRICHOS domains interact with Aβ 40 and Aβ 42 present in neurons and reduce Aβ 42 amyloid fibril formation in vitro , but Bri3 BRICHOS is less efficient. These results indicate that Bri2 and Bri3 BRICHOS have different roles in relation to Aβ aggregation.

Alzheimer’s disease (AD) is the most common form of dementia and there is no successful treatment available. Evidence suggests that fibril formation of the amyloid β-peptide (Aβ) is a major underlying cause of AD, and treatment strategies that reduce the toxic effects of Aβ amyloid are sought for. The BRICHOS domain is found in several proteins, including Bri2 (also called integral membrane protein 2B (ITM2B)), mutants of which are associated with amyloid and neurodegeneration, and Bri3 (ITM2C). We have used mouse hippocampal neurons and brain tissues from mice and humans and show Bri3 deposits dispersed on AD plaques. In contrast to what has been shown for Bri2, Bri3 immunoreactivity is decreased in AD brain homogenates compared to controls. Both Bri2 and Bri3 BRICHOS domains interact with Aβ40 and Aβ42 present in neurons and reduce Aβ42 amyloid fibril formation in vitro, but Bri3 BRICHOS is less efficient. These results indicate that Bri2 and Bri3 BRICHOS have different roles in relation to Aβ aggregation.

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

Background and objective: Cardiovascular diseases are still a major public health concern in Estonia despite the decline in the mortality rate during the past decade. For better preventive strategies we aimed to investigate the prevalence of cardiovascular disease risk factors and their relations with age, gender and ethnicity.Materials and methods: The cross-sectional study was carried out in Tallinn, Estonia. Two hundred individuals from each of the sex and 10-year age group (range 20–65 years of age) were randomly selected and invited to participate. Final study sample consisted of 511 men and 600 women (mean age of 46 years). Physiological measurements were taken and blood samples were drawn for standard measurements of the following markers: total cholesterol, high- and low-density lipoprotein cholesterol, apolipoproteins, triglycerides, glucose and inflammatory markers.Results: Overall, 31% of the study subjects had high blood pressure, 23% had metabolic syndrome, and 55% were overweight/obese. The prevalence of all risk factors increased with age amongst both genders. The proportion of individuals having increased cholesterol, apolipoprotein B-100, and homocysteine levels was very high amongst both genders (60–80%). More Russians and other ethnic minorities compared to ethnic Estonians had calculated 10-year CHD risk ≥ 10%.Conclusions: The study established a high prevalence of cardiovascular disease risk factors in Estonian adults (20–65 years of age). Younger portion of the population and some extent ethnic considerations should be taken into account when designing future studies, health prevention activities and interventions.

Background: This paper will study factors behind leisure sports activity. Physical inactivity is a cause of cardiovascular diseases; therefore, it is important to know the factors related to sporting activity. The study is conducted on a sample from Estonia, which is a country with very high mortality from cardiovascular diseases. Material and methods: The study involved 1184 participants – 639 women (aged 45.7 ±12.4 years) and 545 men (aged 45.5 ±12.1 years). The following information was gathered: leisure sports activity, age, sex, marital status, ethnicity, income, education, body mass index (BMI), smoking, alcohol consumption and physical activity at work. Participants were divided into three groups using a leisure sports activity level: 1) inactive; 2) insufficient sporting activity; 3) at least minimal recommended sporting activity. Results: Statistically significant relationship (p > 0.05) was found between leisure sports activity and ethnicity, education, and income. This relationship is confirmed for men and women. In addition, statistically significant relationship was found between leisure sports activity and age, marital status, smoking, and physical activity at work for men. Conclusions: Policymakers should pay more attention to people who belong to the risk groups of low physical activity.

Background: Although Eastern Europe, including Estonia, has one of the highest morbidity and mortality rates associated with hypertension, there is little information in the literature concerning the biochemical risk factor profile or its association with hypertension in Estonia. This study examined the cross-sectional gender-stratified association between biochemical risk markers and hypertension in a population-based sample of adults in Estonia. Methods: The study was carried out in Tallinn, Estonia and consisted of 511 men and 600 women with a mean age of 46 years. Physiological measurements were taken and blood samples drawn to measure the following markers: cholesterol, high-and low-density lipoprotein cholesterol, apolipoproteins A-1 and B, lipoprotein(a), triglycerides, glucose, fibrinogen, high-sensitivity C-reactive protein and homocysteine. Results: Overall, 36% of participants had hypertension, with approximately 80% being aware of their condition. A total of 40% of participants reported taking antihypertensive medication. Multivariate binary logistic regression analysis showed that a decrease in high-density lipoprotein cholesterol and increases in age, body mass index, apolipoprotein B, triglyceride and homocysteine levels were associated with an increased probability of hypertension. Conclusions: Elevations in biochemical markers and cardiovascular risk factors are associated with hypertension. Increasing body mass index, triglyceride, apolipoprotein B and homocysteine levels with decreasing high-density lipoprotein cholesterol level should be investigated and monitored in Estonian adults.

Despite the fact that homeless people die quite early from heart disease, our study has shown that widely recognized risk factors for cardiovascular disease (CVD) were in the normal endemic range among people with an unhealthy lifestyle. Recently, an increasing number of studies underline the impact of the pathogenetic role of high-grade oxidative stress in the development of different diseases, including CVD. However, corresponding data regarding homeless people is scarce, and therefore, the aim of this pilot study was to investigate the systemic oxidative stress index via the characterization of two opposite sides of the condition. Blood samples were drawn from 46 homeless (age 44±13 years) and 55 healthy (age 47±4 years) male subjects. Total peroxide and total antioxidant responses were determined from the blood serum and an oxidative stress index was calculated. The comparison between the groups revealed that the homeless group had a significantly (p < 0.001) higher oxidative stress index and lower antioxidant marker compared to the control group. Our data demonstrates pathogenetic systemic high-grade oxidative stress in homeless people at the Mustamae District shelter in Tallinn. Considering early diagnostics, prevention and treatment estimations for CVD, it would be worthwhile to conduct a more comprehensive study of oxidative stress markers.