Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning-based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of ß-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid ß and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid ß with hydrophobic surfaces in ß-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen-deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary ß-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

Biological activity versus physiological function of proinsulin C-peptide.

Proinsulin C-peptide (C-peptide) has drawn much research attention. Even if the peptide has turned out not to be important in the treatment of diabetes, every phase of C-peptide research has changed our view on insulin and peptide hormone biology. The first phase revealed that peptide hormones can be subject to processing, and that their pro-forms may involve regulatory stages. The second phase revealed the possibility that one prohormone could harbor more than one activity, and that the additional activities should be taken into account in the development of hormone-based therapies. In the third phase, a combined view of the evolutionary patterns in hormone biology allowed an assessment of C-peptide´s role in physiology, and of how biological activities and physiological functions are shaped by evolutionary processes. In addition to this distinction, C-peptide research has produced further advances. For example, C-peptide fragments are successfully administered in immunotherapy of type I diabetes, and plasma C-peptide levels remain a standard for measurement of beta cell activity in patients. Even if the concept of C-peptide as a hormone is presently not supported, some of its bioactivities continue to influence our understanding of evolutionary changes of also other peptides.

High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry.

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 Aß40 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.

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors.

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

Systemic AA amyloidosis in the red fox (Vulpes vulpes).

Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full-length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter-species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.

Efficient protein production inspired by how spiders make silk.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

Differential hemoglobin A sequestration between hemodialysis modalities.

This report evaluates plasma protein patterns, dialysates and protein analysis of used dialysis membranes from the same patient under hemodialysis in three separate modalities, using high-flux membranes in concentration-driven transport (HD), convection-driven hemofiltration (HF) and combined hemodialfiltration (HDF). The plasma protein changes induced by each of the three dialysis modalities showed small differences in proteins identified towards our previous plasma analyses of chronic kidney disease (CKD) patients. The used dialysate peptide concentrations likewise exhibited small differences among the modalities and varied in the same relative order as the plasma changes, with protein losses in the order HD>HDF>HF. The membrane protein deposits allowed quantification of the relative Hb removal ratios as ~1.7 for HD and ~1.2 for HDF vs. ~1.0 for HF. Hence, plasma protein alterations, dialysate peptide contents and membrane Hb deposits all identify HD as the modality with the most extensive filtration results and exemplifies the accessibility of protein analysis of used membrane filters for evaluation of dialysis efficiencies.

MDR-alcohol dehydrogenases.

Close to 80 years of research have brought MDR alcohol dehydrogenases (ADHs) from unknown molecular concepts to molecules known in exact detail regarding structural, functional and evolutionary properties. They can be traced backwards in at least six stages of development, to essentially the origin of cellular life, and have been monitored in a long series of biannual meetings on "Carbonyl Metabolism". In between each of these latest meetings, a roughly three-fold increase in known totals of MDR databank entries has been apparent, bringing the total now of known MDR-ADH entries to approaching half a million forms.

Specific chaperones and regulatory domains in control of amyloid formation.

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.

Complementary LC-MS/MS Proteomic Analysis of Uremic Plasma Proteins.

OBJECTIVE: To complement an earlier analysis of protein alterations in plasma from uremic versus healthy subjects by addition of further LC-MS/MS analysis to the previously used MALDI-TOF mass analyses. METHODOLOGY: Sequence identifications of tryptic peptides from SDS gel electrophoretic fractions of immunodepleted and HPLC-fractionated plasma was performed from seven chronic kidney disease stage 5 patients (age 55 ± 14 years, glomerular filtration rate 6.9 ±2.9 mL/minute/1.73 m2) and from seven matched controls. RESULTS: About twice as many proteins were increased in uremic plasma as the previously identified. The identifications included proteins that consistently complement the two identification patterns regarding separate subunits from the same protein complex. CONCLUSION: Mass spectrometric analysis is applicable to complex plasma proteomes in clinical settings. The LC-MS/MS technique, based on individual peptide sequence analyses, gives increased identifications and also demonstrates feasibility of this technique in clinical practice.

Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types.

C-peptide evolution: generation from few structural restrictions of bioactivities not necessarily functional.

The proinsulin C-peptide has molecular, cellular and organismal activities but lacks disease-associated mutations or short-term loss-of-function effects. This dilemma between activity and function may be explained from its evolutionary setting with insulin as an ancestral partner. The charge, approximate length and flexibility of C-peptide are all that is required for the insulin interactions, while remaining aspects are free to evolve, where new bioactivities can emerge. They can initially be transient, weak, and non-functional, but may gradually be consolidated. In this manner, C-peptide may have acquired multiple bioactivities, explaining why some yet have limited functions but could represent early-stage hormonal-like activities.

Alcohol dehydrogenase, SDR and MDR structural stages, present update and altered era.

It is now about half a century since molecular research on alcohol dehydrogenase (ADH), short-chain dehydrogenase/reductase (SDR) and medium-chain dehydrogenase/reductase (MDR) started. During this time, at least four stages of research can be distinguished, which led to many ADH, SDR and MDR structures from which their origins could be traced. An introductory summary of these stages is given, followed by a current update on the now known structures, including the present pattern of mammalian MDR-ADH enzymes into six classes and their evolutionary relationships. In spite of the wide spread in evolutionary changes from the "constant" class III to the more "variable" other classes, the change in class V (only confirmed as a transcript in humans) and class VI (absent in humans) are also restricted. Such spread in variability is visible also in other dehydrogenases, but not always so restricted in other co-evolving proteins we have studied. Finally, the shift in era of present ADH research is highlighted, as well as levels of likely future continuation.

The structure, molecular interactions and bioactivities of proinsulin C-peptide correlate with a tripartite molecule.

Many biological roles have been assigned to proinsulin C-peptide over the years. Some appear surprisingly disparate and sometimes even contradictory, like chaperone-like actions and depository tendencies. This review summarizes recently reported biomolecular interactions of the peptide and presents how they correlate with structural and functional aspects into a partitioned molecular architecture. At the structural level, the C-peptide sequence and fold can be subdivided into three distinct parts ('tripartite'). At the functional level, its chaperone-like abilities, self-assembly, and membrane interactions, as well as interactions with relevant proteins can be separately ascribed to these three segments. At the biological level, the assignments are compatible with the suggested roles of C-peptide in granular insulin storage, chaperone-like activities on insulin oligomers, possible depository tendencies, and proposed receptor interactions. Finally, the assignments give interesting parallels to further bioactive peptides, including glucagon and neurotensin. Provided pharmaceutical and clinical trials are successfully completed, the present interpretations should supply mechanistic explanations on C-peptide as a bioactive compound of importance in health and diabetes.

Transthyretin microheterogeneity and molecular interactions: implications for amyloid formation.

Aggregation of transthyretin (TTR), a plasma-binding protein for thyroxine and retinol-binding protein, is the cause of several amyloid diseases. Disease-associated mutations are well known, but wild-type TTR is, to a lesser extent, also amyloidogenic. Monomerization, not oligomer formation as in several other depository diseases, is the rate-limiting step in TTR aggregation, and stabilization of the natively tetrameric form can inhibit amyloid formation. Modifications on Cys10, as well as interactions with native ligands in plasma, were early found to influence the equilibrium between tetrameric and monomeric TTR by dissociating or stabilizing the tetramer. Following these discoveries, synthetic ligands for pharmacological prevention of TTR aggregation could be developed. In this article, we outline how the different types of TTR interactions and its microheterogeneity in plasma are related to its propensity to form amyloid fibrils. We conclude that plasma constituents and dietary components may act as natural TTR stabilizers whose mechanisms of action provide cues for the amelioration of TTR amyloid disease.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive ß-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

A subdivided molecular architecture with separate features and stepwise emergence among proinsulin C-peptides.

The C-peptide of proinsulin exhibits multiple activities and several of the underlying molecular interactions are known. We recently showed that human C-peptide is sub-divided into a tripartite architecture and that the pattern, rather than the exact residue positions, is a characteristic feature. We have now analyzed 75 proinsulins, ranging from fish to human and find a limited co-evolution with insulin, but with many marked deviations. This suggests a complex relationship, in which not only insulin affects the evolution of C-peptide. A subdivided nature, however, is a characteristic feature among all C-peptides, with the N-terminal segment the one most conserved. This segment, ascribed chaperoning charge-interactions with insulin, suggests that the insulin interactions constitute a basic function, although largely shifting from Glu to Asp residues in C-peptides of lower life forms. A second conserved feature is a mid-segment with a high content of adjacent Pro and Gly residues, in mammalian C-peptides compatible with a turn structure, but with fewer and more distantly interspaced such residues in the non-mammalian forms, and even absent in several fish forms. However, this segment of coelacanth C-peptide possesses a unique Cys distribution, capable of forming a disulfide-stabilized turn. Finally, the C-terminal segment of mammalian C-peptides, ascribed a possible receptor-interacting function, is not really discernable in the sub-mammalian forms. Combined, these patterns suggest an evolutionary stepwise acquisition of the tripartite mammalian C-peptide molecule, with insulin-interaction being ancestral, various turn stabilizations apparently of intermediate emergence, and possible receptor-interaction the most recent addition.