The biogenesis of potassium transporters: implications of disease-associated mutations.

The concentration of intracellular and extracellular potassium is tightly regulated due to the action of various ion transporters, channels, and pumps, which reside primarily in the kidney. Yet, potassium transporters and cotransporters play vital roles in all organs and cell types. Perhaps not surprisingly, defects in the biogenesis, function, and/or regulation of these proteins are linked to range of catastrophic human diseases, but to date, few drugs have been approved to treat these maladies. In this review, we discuss the structure, function, and activity of a group of potassium-chloride cotransporters, the KCCs, as well as the related sodium-potassium-chloride cotransporters, the NKCCs. Diseases associated with each of the four KCCs and two NKCCs are also discussed. Particular emphasis is placed on how these complex membrane proteins fold and mature in the endoplasmic reticulum, how non-native forms of the cotransporters are destroyed in the cell, and which cellular factors oversee their maturation and transport to the cell surface. When known, we also outline how the levels and activities of each cotransporter are regulated. Open questions in the field and avenues for future investigations are further outlined.

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

A functional platform for the selection of pathogenic variants of PMM2 amenable to rescue via the use of pharmacological chaperones.

Different strategies are being investigated for treating PMM2-CDG, the most common congenital disorder of glycosylation. The use of pharmacochaperones (PCs) is one of the most promising. The present work characterizes the expression, stability, and enzymatic properties of 15 previously described clinical variants of the PMM2 protein, four novel variants, the Pmm2 mouse variant p.Phe115Leu, and its p.Phe119Leu human counterpart, with the aim of extending the potential use of pharmacochaperoning treatment. PMM2 variants were purified as stable homodimers, except for p.Asp65Gly, p.Ile120Thr, and p.Thr237Lys (no expression detected), p.Thr226Ser and p.Val231Met (aggregates), and p.Glu93Ala, p.Phe119Leu, and p.Phe115Leu (partial dissociated). Enzyme activity analyses identified severe variants and milder ones. Pure dimeric mutant proteins showed a reduction in thermal stability except for p.Asn216Asp. The thermal stability of all the unstable mutants was recovered in the presence of the PC compound VIII. This study adds to the list of destabilizing human variants amenable to rescue by small chemical compounds that increase the stability/activity of PMM2. The proposed platform can be reliably used for assessing the disease-causing effects of PMM2 missense variants, for assessing the correlation between genotype and phenotype, for confirming new clinical defects, and for identifying destabilizing mutations amenable to rescue by PCs.

Bacteria-Derived Protein Aggregates Contribute to the Disruption of Host Proteostasis.

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis-possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut-brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria's effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host-bacteria interactions in the context of protein conformational diseases.

Nanochaperone-Based Strategies to Control Protein Aggregation Linked to Conformational Diseases.

The generation of highly organized amyloid fibrils is associated with a wide range of conformational pathologies, including primarily neurodegenerative diseases. Such disorders are characterized by misfolded proteins that lose their normal physiological roles and acquire toxicity. Recent findings suggest that proteostasis network impairment may be one of the causes leading to the accumulation and spread of amyloids. These observations are certainly contributing to a new focus in anti-amyloid drug design, whose efforts are so far being centered on single-target approaches aimed at inhibiting amyloid aggregation. Chaperones, known to maintain proteostasis, hence represent interesting targets for the development of novel therapeutics owing to their potential protective role against protein misfolding diseases. In this minireview, research on nanoparticles that can either emulate or help molecular chaperones in recognizing and/or correcting protein misfolding is discussed. The nascent concept of "nanochaperone" may indeed set future directions towards the development of cost-effective, disease-modifying drugs to treat several currently fatal disorders.

Expression and purification of amyloid ß-protein, tau, and α-synuclein in Escherichia coli: a review.

Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid ß-protein, tau, and α-synuclein in microbial expression systems, especially Escherichia coli, and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.

Second-Generation Pharmacological Chaperones: Beyond Inhibitors.

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions.

Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism.

In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB.

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.

Rapid and high-throughput colorimetric screening for anti-aggregation reagents of protein conformational diseases by using gold nanoplasmonic particles.

Cellular deposition of destabilized proteins and their aggregates is considered one of the most indisputable factors implicated in protein conformational diseases. Here, we report an innovative high-throughput screening method for discovering anti-aggregation reagents out of numerous potential candidates by using gold nanoplasmonic particles. In our method, nanoparticles act as catalytic activators to accelerate protein aggregation and simultaneously exhibit a colorimetric response according to their embedded shape on the protein aggregates. Using this principle, we observed the colorimetric response to the anti-aggregation effect of amyloid ß (Aß) with the naked eye within a few minutes. Investigation of the anti-aggregation effects of select candidates under three different protein aggregation stages showed that the anti-aggregation efficiency could relate to disease progression. Finally, results obtained with spiked samples in cerebrospinal fluid as well as under various denaturation conditions and different Aß compositions show the feasibility of future personalized medicine considering individual patient's disease progression.

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In†Vivo.

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics.

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

The formation, function and regulation of amyloids: insights from structural biology.

Amyloid diseases are characterized by the accumulation of insoluble, ß-strand-rich aggregates. The underlying structural conversions are closely associated with cellular toxicity, but can also drive the formation of functional protein assemblies. In recent years, studies in the field of structural studies have revealed astonishing insights into the origins, mechanisms and implications of amyloid formation. Notably, high-resolution crystal structures of peptides in amyloid-like fibrils and prefibrillar oligomers have become available despite their challenging chemical nature. Nuclear magnetic resonance spectroscopy has revealed that dynamic local polymorphisms in the benign form of the prion protein affect the transformation into amyloid fibrils and the transmissibility of prion diseases. Studies of the structures and interactions of chaperone proteins help us to understand how the cellular proteostasis network is able to recognize different stages of aberrant protein folding and prevent aggregation. In this review, we will focus on recent developments that connect the different aspects of amyloid biology and discuss how understanding the process of amyloid formation and the associated defence mechanisms can reveal targets for pharmacological intervention that may become the first steps towards clinically viable treatment strategies.

The role of surface electrostatics on the stability, function and regulation of human cystathionine ß-synthase, a complex multidomain and oligomeric protein.

Human cystathionine ß-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.

The interplay between protein stability and dynamics in conformational diseases: the case of hPGK1 deficiency.

Conformational diseases often show defective protein folding efficiency in vivo upon mutation, affecting protein properties such as thermodynamic stability and folding/unfolding/misfolding kinetics as well as the interactions of the protein with the protein homeostasis network. Human phosphoglycerate kinase 1 (hPGK1) deficiency is a rare inherited disease caused by mutations in hPGK1 that lead to loss-of-function. This disease offers an excellent opportunity to explore the complex relationships between protein stability and dynamics because of the different unfolding mechanisms displayed towards chemical and thermal denaturation. This work explores these relationships using two thermostable mutants (p.E252A and p.T378P) causing hPGK1 deficiency and WT hPGK1 using proteolysis and chemical denaturation. p.T378P is degraded ~30-fold faster at low protease concentrations (here, the proteolysis step is rate-limiting) and ~3-fold faster at high protease concentrations (where unfolding kinetics is rate-limiting) than WT and p.E252A, indicating that p.T378P is thermodynamically and kinetically destabilized. Urea denaturation studies support the decrease in thermodynamic stability and folding cooperativity for p.T378P, as well as changes in folding/unfolding kinetics. The present study reveals changes in the folding landscape of hPGK1 upon mutation that may affect protein folding efficiency and stability in vivo, also suggesting that native state stabilizers and protein homeostasis modulators may help to correct folding defects in hPGK1 deficiency. Moreover, detailed kinetic proteolysis studies are shown to be powerful and simple tools to provide deep insight into mutational effects on protein folding and stability in conformational diseases.

Prions: a model of conformational disease?

The discovery that a protein could mimic viral and bacterial pathogens around 1980 by Stanley Prusiner was unexpected. Evidence shows now that Creutzfeldt-Jakob disease and related disorders are caused by prions. Prions and, for example neurodegeneratives diseases, arise from the same general disease mechanism. In each, there is abnormal unfolding and then aggregation of proteins. The protein conformational changes associated with the pathogenesis of protein misfolding disorders produce ß sheet rich oligomers that are partially resistant to proteolysis and have a high tendency to form amyloid-like aggregates. It is important to distinguish between prions and amyloids: prions need not to polymerize into amyloid fibrils and can undergo self-propagation as oligomers. The prion diseases are characterized by the conformational conversion of PrP(c) to PrP(sc), the fundamental even underlying prion diseases. Despite the obvious differences between prions and conventional infectious microorganisms, prions fulfill the Koch's postulates. Meaningful treatments are likely to require cocktails of drugs that interfere with the conversion of precursor into prions and enhance the clearance of prions; such an approach may find application in the more common degenerative diseases.

A novel pathological mutant reveals the role of torsional flexibility in the serpin breach in adoption of an aggregation-prone intermediate.

Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates ('polymers') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation-prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild-type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8-fold faster rate overall. X-ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the 'breach' region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent-exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation.

Pharmacological stabilization of the native state of full-length immunoglobulin light chains to treat light chain amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is a cancer of plasma cells that secrete unstable full-length immunoglobulin light chains. These light chains misfold and aggregate, often with aberrant endoproteolysis, leading to organ toxicity. AL is currently treated by pharmacological elimination of the clonal plasma cells. Since it remains difficult to completely kill these cells in the majority of patients, we seek a complementary drug that inhibits light chain aggregation, which should diminish organ toxicity. We discovered a small-molecule binding site on full-length immunoglobulin light chains by structurally characterizing hit stabilizers emerging from a high-throughput screen seeking small molecules that protect full-length light chains from conformational excursion-linked endoproteolysis. The x-ray crystallographic characterization of 7 structurally distinct hit native-state stabilizers provided a structure-based blueprint, reviewed herein, to design more potent stabilizers. This approach enabled us to transform hits with micromolar affinity into stabilizers with nanomolar dissociation constants that potently prevent light chain aggregation.