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Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.

Naked mole-rats (Heterocephalus glaber) (NMRs) are the longest living rodents known. They show negligible senescence, and are resistant to cancers and certain damaging effects associated with aging. The insulin-like growth factors (IGFs) have pluripotent actions, influencing growth processes in virtually every system of the body. They are established contributors to the aging process, confirmed by the demonstration that decreased IGF signaling results in life-extending effects in a variety of species. The IGFs are likewise involved in progression of cancers by mediating survival signals in malignant cells. This report presents a full characterization of the IGF system in the NMR: ligands, receptors, IGF binding proteins (IGFBPs), and IGFBP proteases. A particular emphasis was placed on the IGFBP protease, pregnancy-associated plasma protein-A (PAPP-A), shown to be an important lifespan modulator in mice. Comparisons of IGF-related genes in the NMR with human and murine sequences indicated no major differences in essential parts of the IGF system, including PAPP-A. The protease was shown to possess an intact active site despite the report of a contradictory genome sequence. Furthermore, PAPP-A was expressed and translated in NMRs cells and retained IGF-dependent proteolytic activity towards IGFBP-4 and IGF-independent activity towards IGFBP-5. However, experimental data suggest differential regulatory mechanisms for PAPP-A expression in NMRs than those described in humans and mice. This overall description of the IGF system in the NMR represents an initial step towards elucidating the complex molecular mechanisms underlying longevity, and how these animals have evolved to ensure a delayed and healthy aging process.

The aim of this study was to investigate the potential of potato proteins and peptides as emulsifiers in the microencapsulation of fish oil by spray-drying. Microcapsules were produced using a potato protein extract, and fractions enriched in patatin and protease inhibitors. Furthermore, bioinformatically predicted emulsifier peptides from abundant potato proteins and a hydrolysate, obtained through targeted proteolysis of the extract, were investigated. During 28 days of storage at 25 °C, peptides and the hydrolysate exhibited better emulsifying properties and higher encapsulation efficiencies compared to native proteins and sodium caseinate. Significant differences (p < 0.05) were observed in the peroxide value (PV) and secondary volatile oxidation products between the microcapsules produced with peptides and native proteins. Microcapsules produced with peptides and hydrolysate showed the highest oxidative stability, not exceeding a PV of 10 meq/kg oil, and with concentrations of volatiles below the odor threshold in oil for five of the six studied compounds. These results show the emulsifying potential of potato peptides and hydrolysate for use in microencapsulation of hydrophobic bioactive ingredients such as fish oil.

Abstract Brown bears hibernate throughout half of the year as a survival strategy to reduce energy consumption during prolonged periods with scarcity of food and water. Thyroid hormones are the major endocrine regulators of basal metabolic rate in humans. Therefore, we aimed to determine regulations in serum thyroid hormone levels in hibernation compared to the active state to investigate if these are involved in the adaptions for hibernation. We used electrochemiluminescence immunoassay to quantify total triiodothyronine (T3) and thyroxine (T4) levels in hibernation and active state in paired serum samples from six subadult Scandinavian brown bears. Additionally, we determined regulations in the liver mRNA levels of three major thyroid hormone-binding proteins; thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin, by analysis of previously published grizzly bear RNA sequencing data. We found that bears were hypothyroid when hibernating with T4 levels reduced to less than 44% (P = 0.008) and T3 levels reduced to less than 36% (P = 0.016) of those measured in the active state. In hibernation, mRNA levels of TBG and albumin increased to 449% (P = 0.031) and 121% (P = 0.031), respectively, of those measured in the active state. TTR mRNA levels did not change. Hibernating bears are hypothyroid and share physiologic features with hypothyroid humans, including decreased basal metabolic rate, bradycardia, hypothermia, and fatigue. We speculate that decreased thyroid hormone signaling is a key mediator of hibernation physiology in bears. Our findings shed light on the translational potential of bear hibernation physiology to humans for whom a similar hypometabolic state could be of interest in specific conditions.

Abstract Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Sex hormone‐binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell‐based expression system for recombinant full‐length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10‐fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~ 58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in the presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [3H]DHT were determined to 0.21 ± 0.04 nm for human and 1.32 ± 0.10 nm for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology.

In this article, we present mass-spectrometry based plasma proteomics data from hibernating and active free-ranging Scandinavian brown bears (Ursus arctos).The brown bear hibernates for half the year. Despite obesity when entering the den and the prolonged period of inactivity, the bear shows no signs of the harmful effects associated with these conditions in humans. Thus, the hibernating bear is a potential translational model for addressing these complications in humans.We analyzed plasma samples from fourteen 2- to 3-year-old bears (6 males and 8 females) collected both during hibernation and the active state, and for some of the bears during two seasons, resulting in a total of 38 analyzed plasma samples.In triplicates, the plasma proteins were unfolded and reduced. To increase the chance of detecting low-molecular-weight proteins and peptides, we filtered the samples using a 50 K molecular weight cut-off filter with the aim to deplete larger abundant proteins, including albumin, and thereby increase the depth of the analysis. The proteins in the permeate were then tryptically digested, desalted, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed with the MaxQuant software searching against an Ursus arctos horribilis protein database.Here, we provide the raw data, a list with identified proteins in the plasma samples, and the databases applied for protein identification.Based on the provided data, differentially expressed proteins in hibernation compared to active state can be identified. These proteins may be involved in the bears’ adaptions to hibernation physiology and hold potential as novel therapeutic targets.

Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.