Aggregation Of Target Proteins Research Articles

Target enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying

Anhydrobiotic organisms accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered proteins (IDPs) reported to improve cellular tolerance to water stress. Here we show that AfrLEA6, a Group 6 LEA protein only recently discovered in animals, protects lactate dehydrogenase (LDH), citrate synthase (CS) and phosphofructokinase (PFK) against damage during desiccation. In some cases, protection is enhanced by trehalose, a naturally-occurring protective solute. An open question is whether gain of secondary structure by LEA proteins during drying is a prerequisite for this stabilizing function. We used incremental drying (equilibration to a series of relative humidities, RH) to test the ability of AfrLEA2, a Group 3 LEA protein, to protect desiccation-sensitive PFK. AfrLEA2 was chosen due to its exceptional ability to protect PFK. In parallel, circular dichroism (CD) spectra were obtained for AfrLEA2 across the identical range of relative water contents. Protection of PFK by AfrLEA2, above that observed with trehalose and BSA, coincides with simultaneous gain of α-helix in AfrLEA2. At 100% RH, the CD spectrum for AfrLEA2 is typical of random coil, while at decreasing RH, the spectrum shows higher ellipticity at 191 nm and minima at 208 and 220 nm, diagnostic of α-helix. This study provides experimental evidence linking the gain of α-helix with stabilization of a target protein across a graded series of hydration states. Mechanistically, it is intriguing that certain other functions of these IDPs, like preventing aggregation of target proteins, can occur in fully hydrated cells and apparently do not require gain of α-helix.

Therapeutic Approaches Targeting Protein Aggregation in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease that targets motor neurons (MNs) in the brain and spinal cord. It leads to gradual loss of motor signals to muscles leading to atrophy and weakness. Most patients do not survive for more than 3–5 years after disease onset. Current ALS treatments provide only a small delay of disease progression. Therefore, it is of utmost importance to explore new therapeutic approaches. One of the major hindrances in achieving this goal is poor understanding of causes of the disease. ALS has complex pathophysiological mechanisms in its genetic and sporadic forms. Protein aggregates are a common hallmark of ALS regardless of cause making protein pathways attractive therapeutic targets in ALS. Here, we provide an overview of compounds in different stages of pharmacological development and their protein pathway targets.

The multifaceted nature of αB-crystallin.

In vivo, small heat-shock proteins (sHsps) are key players in maintaining a healthy proteome. αB-crystallin (αB-c) or HspB5 is one of the most widespread and populous of the ten human sHsps. Intracellularly, αB-c acts via its molecular chaperone action as the first line of defence in preventing target protein unfolding and aggregation under conditions of cellular stress. In this review, we explore how the structure of αB-c confers its function and interactions within its oligomeric self, with other sHsps, and with aggregation-prone target proteins. Firstly, the interaction between the two highly conserved regions of αB-c, the central α-crystallin domain and the C-terminal IXI motif and how this regulates αB-c chaperone activity are explored. Secondly, subunit exchange is rationalised as an integral structural and functional feature of αB-c. Thirdly, it is argued that monomeric αB-c may be its most chaperone-species active, but at the cost of increased hydrophobicity and instability. Fourthly, the reasons why hetero-oligomerisation of αB-c with other sHsps is important in regulating cellular proteostasis are examined. Finally, the interaction of αB-c with aggregation-prone, partially folded target proteins is discussed. Overall, this paper highlights the remarkably diverse capabilities of αB-c as a caretaker of the cell.

Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism.

Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.

Novel approaches to counter protein aggregation pathology in Parkinson's disease.

The primary neuropathological characteristics of the Parkinsonian brain are the loss of nigral dopamine neurons and the aggregation of alpha synuclein protein. Efforts to development potentially disease-modifying treatments have largely focused on correcting these aspects of the condition. In the last decade treatments targeting protein aggregation have entered the clinical pipeline. In this chapter we provide an overview of ongoing clinical trial programs for different therapies attempting to reduce protein aggregation pathology in Parkinson's disease. We will also briefly consider various novel approaches being proposed-and being developed preclinically-to inhibit/reduce aggregated protein pathology in Parkinson's.

Advances in the Prediction of Protein Aggregation Propensity.

Protein aggregation into β-sheet-enriched insoluble assemblies is being found to be associated with an increasing number of debilitating human pathologies, such as Alzheimer's disease or type 2 diabetes, but also with premature aging. Furthermore, protein aggregation represents a major bottleneck in the production and marketing of proteinbased therapeutics. Thus, the development of methods to accurately forecast the aggregation propensity of a certain protein is of much value. A myriad of in vitro and in vivo aggregation studies have shown that the aggregation propensity of a certain polypeptide sequence is highly dependent on its intrinsic properties and, in most cases, driven by specific short regions of high aggregation propensity. These observations have fostered the development of a first generation of algorithms aimed to predict protein aggregation propensities from the protein sequence. A second generation of programs able to map protein aggregation on protein structures is emerging. Herein, we review the most representative online accessible predictive tools, emphasizing their main distinctive features and the range of applications. In this review, we describe representative biocomputational approaches to evaluate the aggregation properties of protein sequences and structures, while illustrating how they can become very useful tools to target protein aggregation in biomedicine and biotechnology.

A Split Transcriptional Repressor That Links Protein Solubility to an Orthogonal Genetic Circuit.

Monitoring the aggregation of proteins within the cellular environment is key to investigating the molecular mechanisms underlying the formation of off-pathway protein assemblies associated with the development of disease and testing therapeutic strategies to prevent the accumulation of non-native conformations. It remains challenging, however, to couple protein aggregation events underlying the cellular pathogenesis of a disease to genetic circuits and monitor their progression in a quantitative fashion using synthetic biology tools. To link the aggregation propensity of a target protein to the expression of an easily detectable reporter, we investigated the use of a transcriptional AND gate system based on complementation of a split transcription factor. We first identified two-fragment tetracycline repressor (TetR) variants that can be regulated via ligand-dependent induction and demonstrated that split TetR variants can function as transcriptional AND gates in both bacteria and mammalian cells. We then adapted split TetR for use as an aggregation sensor. Protein aggregation was detected by monitoring complementation between a larger TetR fragment that serves as a "detector" and a smaller TetR fragment expressed as a fusion to an aggregation-prone protein that serves as a "sensor" of the target protein aggregation status. This split TetR represents a novel genetic component that can be used for a wide range of applications in bacterial as well as mammalian synthetic biology and a much needed cell-based sensor for monitoring a protein's conformational status in complex cellular environments.

Novel Application of Magnetic Protein: Convenient One-Step Purification and Immobilization of Proteins

Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the magnetic protein was firstly used in biotechnology application, and a novel convenient one-step purification and immobilization method was established. A universal vector and three linker patterns were developed for fusion expression of magnetic protein and target protein. The magnetic protein was absorbed by iron beads, followed by target protein aggregation, purification, and immobilization. GFP, employed as a reporter protein, was successfully purified from cell lysate. Subsequently, three enzymes (lipase, α-L-arabinofuranosidase, pullulanase) with different molecular sizes testified the versatility of this magnetic-based approach. The specific activities of the purified enzymes were distinctly higher than those of the traditionally purified enzymes using affinity chromatography. The lipase immobilized on iron beads presented improved thermostability and enhanced pH tolerance compared to the free enzyme. The immobilized lipase could be easily recovered and reused for maximum utilization. After 20 cycles of reutilization, the magnetically immobilized lipase retained 71% of its initial activity. This investigation may help introduce magnetic protein into biotechnology applications, and the one-step purification and immobilization method may serve to illustrate an economically viable process for industry.

Selective Knockdowns in Maize by Sequence-Specific Protein Aggregation.

Protein aggregation is determined by 5-15 amino acids peptides of the target protein sequence, so-called aggregation-prone regions (APRs) that specifically self-associate to form β-structured inclusions. The presence of APRs in a target protein can be predicted by a dedicated algorithm, such as TANGO. Synthetic aggregation-prone proteins are designed by expressing specific APRs fused to a fluorescent carrier for stability and visualization. Previously, the stable expression of these proteins in Zea mays (maize) has been demonstrated to induce aggregation of target proteins with specific localization, such as the starch-degrading enzyme α-glucan water dikinase, giving rise to plants displaying knockdown phenotypes. Here, we describe how to design synthetic aggregation-prone proteins to harness the sequence specificity of APRs to generate aggregation-associated phenotypes in a targeted manner and in different subcellular compartments. This method points toward the application of induced targeted aggregation as a useful tool to knock down protein functions in maize and to generate crops with improved traits.

Mannich base approach to 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone: A water-soluble prodrug for a multitarget inhibition of cholinesterases, beta-amyloid fibrillization and oligomer-induced cytotoxicity

Targeting protein aggregation for the therapy of neurodegenerative diseases remains elusive for medicinal chemists, despite a number of small molecules known to interfere in amyloidogenesis, particularly of amyloid beta (Aβ) protein. Starting from previous findings in the antiaggregating activity of a class of indolin-2-ones inhibiting Aβ fibrillization, 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone 1 was identified as a multitarget inhibitor of Aβ aggregation and cholinesterases with IC50s in the low μM range. With the aim of increasing aqueous solubility, a Mannich-base functionalization led to the synthesis of N-methylpiperazine derivative 2. At acidic pH, an outstanding solubility increase of 2 over the parent compound 1 was proved through a turbidimetric method. HPLC analysis revealed an improved stability of the Mannich base 2 at pH2 along with a rapid release of 1 in human serum as well as an outstanding hydrolytic stability of the parent hydrazone. Coincubation of Aβ1–42 with 2 resulted in the accumulation of low MW oligomers, as detected with PICUP assay. Cell assays on SH-SY5Y cells revealed that 2 exerts strong cytoprotective effects in both cell viability and radical quenching assays, mainly related to its active metabolite 1. These findings show that 2 drives the formation of non-toxic, off-pathway Aβ oligomers unable to trigger the amyloid cascade and toxicity.

Methods to Monitor and Quantify Autophagy in the Social Amoeba Dictyostelium discoideum.

Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages from initiation to final degradation of cargo are finely regulated. Therefore, the study of this process requires the use of a palette of techniques, which are continuously evolving and whose interpretation is not trivial. Here, we present the social amoeba Dictyostelium discoideum as a relevant model to study autophagy. Several methods have been developed based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophagy genes and proteins, and examination of the autophagic flux with various techniques. In this review, we discuss the pros and cons of the currently available techniques to assess autophagy in this organism.

Differential modulation of the chaperone-like activity of HSP-1/2, a major protein of horse seminal plasma by anionic and cationic surfactants

The major protein of equine seminal plasma, HSP-1/2 exhibits chaperone-like activity (CLA) by protecting various target proteins against thermal, chemical and oxidative stress. Polydispersity and surface hydrophobicity of HSP-1/2 were found to be important for its CLA. Surfactants are known to alter certain properties of proteins, e.g. hydrophobicity, charge and conformation either by altering properties of the medium or by direct binding. In the current study, thermal aggregation of alcohol dehydrogenase (ADH) and enolase has been studied in the presence of HSP-1/2, different surfactants and their combinations. The results obtained show that anionic surfactants (SDS, sodium dodecyl benzene sulfate) and neutral surfactants (tween-20, triton X-100) increase the CLA of HSP-1/2 and also inhibit aggregation of the target proteins independently. On the other hand, cationic surfactants (CTAB, alanine palmityl ester) increased the thermal aggregation of ADH and enolase and also decreased the CLA of HSP-1/2. These results are of significant interest as they show that surfactants such as SDS and tween-20 can potentially be used as anti-aggregation agents to prevent thermal aggregation of target proteins.

Ubiquitin-Dependent And Independent Signals In Selective Autophagy.

Selective autophagy regulates the abundance of specific cellular components via a specialized arsenal of factors, termed autophagy receptors, that target protein complexes, aggregates, and whole organelles into lysosomes. Autophagy receptors bind to LC3/GABARAP proteins on phagophore and autophagosome membranes, and recognize signals on cargoes to deliver them to autophagy. Ubiquitin (Ub), a well-known signal for the degradation of polypeptides in the proteasome, also plays an important role in the recognition of cargoes destined for selective autophagy. In addition, a variety of cargoes are committed to selective autophagy pathways by Ub-independent mechanisms employing protein-protein interaction motifs, Ub-like modifiers, and sugar- or lipid-based signals. In this article we summarize Ub-dependent and independent selective autophagy pathways, and discuss regulatory mechanisms and challenges for future studies.

Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology

The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in “inclusion bodies” (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ–K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24°C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0–7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3×107IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.

A Hot-Segment-Based Approach for the Design of Cross-Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self-Assembly.

The design of inhibitors of protein-protein interactions mediating amyloid self-assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self-assembly. Here we present a hot-segment-linking approach to design a series of mimics of the IAPP cross-amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self- and cross-seeded IAPP self-assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer's disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well.

Targeting protein aggregation for the treatment of degenerative diseases.

The aggregation of specific proteins is hypothesized to underlie several degenerative diseases, which are collectively known as amyloid disorders. However, the mechanistic connection between the process of protein aggregation and tissue degeneration is not yet fully understood. Here, we review current and emerging strategies to ameliorate aggregation-associated degenerative disorders, with a focus on disease-modifying strategies that prevent the formation of and/or eliminate protein aggregates. Persuasive pharmacological and genetic evidence now supports protein aggregation as the cause of postmitotic tissue dysfunction or loss. However, a more detailed understanding of the factors that trigger and sustain aggregate formation and of the structure-activity relationships underlying proteotoxicity is needed to develop future disease-modifying therapies.

Detection of p62 on Paraffin Sections by Immunohistochemistry.

The study of autophagy in human disease is a rapidly expanding field. Diagnostic paraffin sections of a variety of patient tissues, including bone marrow, are available to researchers-yet are unsuitable for traditional autophagy quantification methods such as western blot or electron microscopy. This protocol outlines the immunohistochemical detection of the protein p62 (sequestosome-1, encoded by the gene SQSTM1)-an indicator of autophagic degradative activity-in slide-mounted paraffin sections such as bone marrow samples cut by a trephine. The p62 protein is an autophagic cargo adaptor, capable of binding to ubiquitylated proteins as well as autophagosome membrane proteins (LC3B and GABA(A) receptor-associated protein [GABARAP] family members) and hypothesized thus to target protein aggregates for lysosomal degradation. p62 itself is degraded by autophagy, remaining at low levels when autophagy is induced, and has been shown to accumulate when autophagy is deficient. Qualitative assessment and comparison of p62 staining between healthy and disease sections or disease subtypes will help target further investigation into the potential roles for autophagy in a variety of disorders.

Effect of crowding on several stages of protein aggregation in test systems in the presence of α-crystallin

Macromolecular crowding can facilitate protein–protein interactions in the cell, in particular aggregation processes. To characterize the anti-aggregation activity of chaperones under conditions mimicking the crowded environment in the cell, two basic test systems are used. Test systems of the first type are based on aggregation of target proteins undergoing unfolding under different factors. Dithithreitol-induced aggregation of α-lactalbumin is used as such a system. The increase in the duration of lag phase after the addition of the crowder (polyethylene glycol; PEG) to the system containing α-crystallin has been interpreted as a retardation of the stages that are the rate-limiting stages of the general process of aggregation (the nucleation stage and the stages of clusterization of nuclei). Test systems of the second type are based on aggregation of UV-irradiated proteins. Such test systems permit investigating the effects of different agents directly on the stages of aggregation of unfolded protein. UV-irradiated glycogen phosphorylase b (Phb) is used as a target protein. Analysis of the initial rate of aggregation after the addition of PEG at different points in time to the mixture of UV-irradiated Phb and α-crystallin allowed estimating the time of half-conversion for the structural rearrangement of the primary UV-irradiated Phb–α-crystallin complex.

Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI).

Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOFI imaging with high spatial-temporal resolution.

Phosphomimics Destabilize Hsp27 Oligomeric Assemblies and Enhance Chaperone Activity

Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27.