Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.     

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62⁻ total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.     

Novel Antibacterial Targets in Protein Biogenesis Pathways

Antibiotic resistance has emerged as a global threat due to the ability of bacteria to quickly evolve in response to the selection pressure induced by anti-infective drugs. Thus, there is an urgent need to develop new antibiotics against resistant bacteria. In this review, we discuss pathways involving bacterial protein biogenesis as attractive antibacterial targets since many of them are essential for bacterial survival and virulence. We discuss the structural understanding of various components associated with bacterial protein biogenesis, which in turn can be utilized for rational antibiotic design. We highlight efforts made towards developing inhibitors of these pathways with insights into future possibilities and challenges. We also briefly discuss other potential targets related to protein biogenesis.     

Inhibition of Amyloid Protein Aggregation Using Selected Peptidomimetics

Aberrant protein aggregation leads to the formation of amyloid fibrils. This phenomenon is linked to the development of more than 40 irremediable diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and cancer. Plenty of research efforts have been given to understanding the underlying mechanism of protein aggregation, associated toxicity, and the development of amyloid inhibitors. Recently, the peptidomimetic approach has emerged as a potential tool to modulate several protein-protein interactions (PPIs). In this review, we discussed selected peptidomimetic-based approaches for the modulation of important amyloid proteins (Islet Amyloid Polypeptide, Amyloid Beta, α-synuclein, mutant p53, and insulin) aggregation. This approach holds a powerful platform for creating an essential stepping stone for the vital development of anti-amyloid therapeutic agents.     

The Role of Crowded Physiological Environments in Prion and Prion-like Protein Aggregation

Prion diseases and prion- like protein misfolding diseases are related to the accumulation of abnormal aggregates of the normal host proteins including prion proteins and Tau protein. These proteins possess self-templating and transmissible characteristics. The crowded physiological environments where the aggregation of these amyloidogenic proteins takes place can be imitated in vitro by the addition of macromolecular crowding agents such as inert polysaccharides. In this review, we summarize the aggregation of prion proteins in crowded physiological environments and discuss the role of macromolecular crowding in prion protein aggregation. We also summarize the aggregation of prion- like proteins including human Tau protein, human α-synuclein, and human copper, zinc superoxide dismutase under macromolecular crowding environments and discuss the role of macromolecular crowding in prion- like protein aggregation. The excluded-volume effects caused by macromolecular crowding could accelerate the aggregation of neurodegenerative disease-associated proteins while inhibiting the aggregation of the proteins that are not neurodegenerative disease-associated.     

Molecular Investigations of Protein Aggregation in the Pathogenesis of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disorder characterized by selective loss of lower and upper motor neurons (MNs) in the brain and spinal cord, resulting in paralysis and eventually death due to respiratory insufficiency. Although the fundamental physiological mechanisms underlying ALS are not completely understood, the key neuropathological hallmarks of ALS pathology are the aggregation and accumulation of ubiquitinated protein inclusions within the cytoplasm of degenerating MNs. Herein, we discuss recent insights into the molecular mechanisms that lead to the accumulation of protein aggregates in ALS. This will contribute to a better understanding of the pathophysiology of the disease and may open novel avenues for the development of therapeutic strategies.     

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable β-sheet enriched intermediates, which are stabilized by intermolecular interactions with other β-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.     

Design of Protein–Protein Interaction Inhibitors Based on Protein Epitope Mimetics

On the scaffold : Grafting protein epitopes onto conformationally designed scaffolds represents a very promising approach to protein ligand design, with potential applications in many areas of chemical biology and molecular medicine. The highlighted article describes the design and anticancer activity of novel p53/MDM2 inhibitors built upon a scorpion toxin scaffold. Other approaches to protein epitope mimetics are also briefly discussed.

     

CRISPR Approaches for the Diagnosis of Human Diseases

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.     

Breaking the Fourth Wall: Modulating Quaternary Associations for Protein Regulation and Drug Discovery

Protein–protein interactions (PPIs) are an effective means to orchestrate intricate biological processes required to sustain life. Approximately 650 000 PPIs underlie the human interactome; thus underscoring its complexity and the manifold signaling outputs altered in response to changes in specific PPIs. This minireview illustrates the growing arsenal of PPI assemblies and offers insights into how these varied PPI regulatory modalities are relevant to customized drug discovery, with a focus on cancer. First, known and emerging PPIs and PPI-targeted drugs of both natural and synthetic origin are categorized. Building on these discussions, the merits of PPI-guided therapeutics over traditional drug design are discussed. Finally, a compare-and-contrast section for different PPI blockers, with gain-of-function PPI interventions, such as PROTACS, is provided.     

Chemical Biology Toolbox to Visualize Protein Aggregation in Live Cells

Protein misfolding and aggregation is a complex biochemical process and has been associated with numerous human degenerative diseases. Developing novel chemical and biological tools and approaches to visualize aggregated proteins in live cells is in high demand for mechanistic studies, diagnostics, and therapeutics. In this review, we summarize the recent developments in the chemical biology toolbox applied to protein aggregation studies in live cells. These methods exploited fluorescent protein tags, fluorescent chemical tags, and small-molecule probes to visualize the protein-aggregation process, detect proteome stresses, and quantify the protein homeostasis network capacity. Inspired by these seminal works, we have generalized design principles for the development of new detection methods and probes in the future that will illuminate this important biological process.     

Mechanism of Suppression of Protein Aggregation by ��-Crystallin

This review summarizes experimental data illuminating the mechanism of suppression of heat-induced protein aggregation by α-crystallin, one of the small heat shock proteins. The dynamic light scattering data show that the initial stage of thermal aggregation of proteins is the formation of the initial aggregates involving hundreds of molecules of the denatured protein. Further sticking of the starting aggregates proceeds in a regime of diffusion-limited cluster-cluster aggregation. The protective effect of α-crystallin is due to transition of the aggregation process to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower than unity.     

Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.     

Heat Shock Protein 90 as Therapeutic Target for CVDs and Heart Ageing

Cardiovascular diseases (CVDs) are the leading cause of death globally, representing approximately 32% of all deaths worldwide. Molecular chaperones are involved in heart protection against stresses and age-mediated accumulation of toxic misfolded proteins by regulation of the protein synthesis/degradation balance and refolding of misfolded proteins, thus supporting the high metabolic demand of the heart cells. Heat shock protein 90 (HSP90) is one of the main cardioprotective chaperones, represented by cytosolic HSP90a and HSP90b, mitochondrial TRAP1 and ER-localised Grp94 isoforms. Currently, the main way to study the functional role of HSPs is the application of HSP inhibitors, which could have a different way of action. In this review, we discussed the recently investigated role of HSP90 proteins in cardioprotection, atherosclerosis, CVDs development and the involvements of HSP90 clients in the activation of different molecular pathways and signalling mechanisms, related to heart ageing.     

Aggregation Condition–Structure Relationship of Mouse Prion Protein Fibrils

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.     

Identification of Natural Products as Potential Pharmacological Chaperones for Protein Misfolding Diseases

Defective protein folding and accumulation of misfolded proteins is associated with neurodegenerative, cardiovascular, secretory, and metabolic disorders. Efforts are being made to identify small-molecule modulators or structural-correctors for conformationally destabilized proteins implicated in various protein aggregation diseases. Using a metastable-reporter-based primary screen, we evaluated pharmacological chaperone activity of a diverse class of natural products. We found that a flavonoid glycoside ( C-10 , chrysoeriol-7-O-β-D-glucopyranoside) stabilizes metastable proteins, prevents its aggregation, and remodels the oligomers into protease-sensitive species. Data was corroborated with additional secondary screen with disease-specific pathogenic protein. In vitro and cell-based experiments showed that C-10 inhibits α-synuclein aggregation which is implicated in synucleinopathies-related neurodegeneration. C-10 interferes in its structural transition into β-sheeted fibrils and mitigates α-synuclein aggregation-associated cytotoxic effects. Computational modeling suggests that C-10 binds to unique sites in α-synuclein which may interfere in its aggregation amplification. These findings open an avenue for comprehensive SAR development for flavonoid glycosides as pharmacological chaperones for metastable and aggregation-prone proteins implicated in protein conformational diseases.     

Aggregation and Precipitation Kinetics of Canola Protein by isoelectric precipitation

Kinetics of aggregation, nucleation and growth of canola protein particles were developed in a 100 mL mixed-suspension mixed-product removal precipitator (MSMPRP) using isoelectric precipitation with HCI at pH 4.2. Protein yield in terms of total nitrogen was measured by Kjeldahl system. The size distribution of protein aggregates was determined by a Coulter counter. The effects of pH, protein concentration driving force, mean residence time, agitation rate, ionic strength of the solution, and the operating temperature on the size distribution and yield of protein particles were investigated. Population balance on the protein particles was used to derive empirical correlations for the index of aggregation, nucleation rate of primary particles and growth rate of protein aggregates.