Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction.

The N-end rule pathway regulates ER stress-induced clusterin release to the cytosol where it directs misfolded proteins for degradation.

Previous work suggests that cell stress induces release of the normally secreted chaperone clusterin (CLU) into the cytosol. We analyzed the localization of CLU in healthy and stressed cells, the mechanism of its cytosolic release, and its interactions with cytosolic misfolded proteins. Key results of this study are the following: (1) full-length CLU is released to the cytosol during stress, (2) the CLU N-terminal D1 residue is recognized by the N-end rule pathway and together with the enzyme ATE1 is essential for cytosolic release, (3) CLU can form stable complexes with cytosolic misfolded proteins and direct them to the proteasome and autophagosomes, and (4) cytosolic CLU protects cells from hypoxic stress and the cytosolic overexpression of an aggregation-prone protein.

Elucidating the Stoichiometries of Host-Pathogen Protein Interactions with Mass Photometry.

Mass photometry (MP) is a single molecule technique that enables the characterization of individual proteins. Here we show a detailed workflow using the Refeyn One to investigate molecular complexes, using the M53 protein, a plasminogen-binding group A streptococcal M-like protein (PAM), and human plasminogen as exemplar proteins. The methodology described herein confirmed a 1:1 binding stoichiometry for the M53-plasminogen complex.

Extracellular protein homeostasis in neurodegenerative diseases.

The protein homeostasis (proteostasis) system encompasses the cellular processes that regulate protein synthesis, folding, concentration, trafficking and degradation. In the case of intracellular proteostasis, the identity and nature of these processes have been extensively studied and are relatively well known. By contrast, the mechanisms of extracellular proteostasis are yet to be fully elucidated, although evidence is accumulating that their age-related progressive impairment might contribute to neuronal death in neurodegenerative diseases.

A Comparative Analysis of Multidimensional COVID-19 Poverty Determinants: An Observational Machine Learning Approach.

Poverty is a glaring issue in the twenty-first century, even after concerted efforts of organizations to eliminate the same. Predicting poverty using machine learning can offer practical models for facilitating the process of elimination of poverty. This paper uses Multidimensional Poverty Index Data from the Oxford Poverty and Human Development Initiative across the years 2019 and 2021 to make predictions of multidimensional poverty before and during the pandemic.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye.

The Emerging Roles of Extracellular Chaperones in Complement Regulation.

The immune system is essential to protect organisms from internal and external threats. The rapidly acting, non-specific innate immune system includes complement, which initiates an inflammatory cascade and can form pores in the membranes of target cells to induce cell lysis. Regulation of protein homeostasis (proteostasis) is essential for normal cellular and organismal function, and has been implicated in processes controlling immunity and infection.

Roles of constitutively secreted extracellular chaperones in neuronal cell repair and regeneration.

Protein quality control involves many processes that jointly act to regulate the expression, localization, turnover, and degradation of proteins, and has been highlighted in recent studies as critical to the differentiation of stem cells during regeneration. The roles of constitutively secreted extracellular chaperones in neuronal injury and disease are poorly understood. Extracellular chaperones are multifunctional proteins expressed by many cell types, including those of the nervous system, known to facilitate protein quality control processes.

N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting Aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein.

Identifying new molecular players in extracellular proteostasis.

Proteostasis refers to a delicately tuned balance between the processes of protein synthesis, folding, localization, and the degradation of proteins found inside and outside cells. Our understanding of extracellular proteostasis is rather limited and largely restricted to knowledge of 11 currently established extracellular chaperones (ECs). This review will briefly outline what is known of the established ECs, before moving on to discuss experimental strategies used to identify new members of this growing family, and an examination of a group of putative new ECs identified using one of these approaches.

Clusterin, other extracellular chaperones, and eye disease.

Proteostasis refers to all the processes that maintain the correct expression level, location, folding and turnover of proteins, essential to organismal survival. Both inside cells and in body fluids, molecular chaperones play key roles in maintaining proteostasis. In this article, we focus on clusterin, the first-recognized extracellular mammalian chaperone, and its role in diseases of the eye.

Neuroserpin and transthyretin are extracellular chaperones that preferentially inhibit amyloid formation.

Neuroserpin is a secreted protease inhibitor known to inhibit amyloid formation by the Alzheimer’s beta peptide (Aβ). To test whether this effect was constrained to Aβ, we used a range of in vitro assays to demonstrate that neuroserpin inhibits amyloid formation by several different proteins and protects against the associated cytotoxicity but, unlike other known chaperones, has a poor ability to inhibit amorphous protein aggregation. Collectively, these results suggest that neuroserpin has an unusual chaperone selectivity for intermediates on the amyloid-forming pathway.

Expanding the family of extracellular chaperones: Identification of human plasma proteins with chaperone activity.

Proteostasis, the balance of protein synthesis, folding and degradation, is essential to maintain cellular function and viability, and the many known intracellular chaperones are recognized as playing key roles in sustaining life. In contrast, the identity of constitutively secreted extracellular chaperones (ECs) and their physiological roles in extracellular proteostasis is less completely understood. We designed and implemented a novel strategy, based on the well-known propensity of chaperones to bind to regions of hydrophobicity exposed on misfolding proteins, to discover new ECs present in human blood.

The Dual Roles of Clusterin in Extracellular and Intracellular Proteostasis.

Clusterin (CLU) was the first reported secreted mammalian chaperone and impacts on serious diseases associated with inappropriate extracellular protein aggregation. Many studies have described intracellular CLU in locations outside the secretory system and recent work has shown that CLU can be released into the cytosol during cell stress. In this article, we critically evaluate evidence relevant to the proposed origins of cellular CLU found outside the secretory system, and advance the hypothesis that the cytosolic release of CLU induced by stress serves to facilitate the trafficking of misfolded proteins to the proteasome and autophagy for degradation.

Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants.

The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Recent independent studies are now exploring the potential use of clusterin as a therapeutic in a variety of disease contexts. In the past, the extensive post-translational processing of clusterin, coupled with its potent binding to essentially any misfolded protein, have meant that its expression as a fully functional recombinant protein has been very difficult.

MicroRNAs as Key Regulators of Ovarian Cancers.

The tumor microenvironment can be realistically viewed as an active battle ground between the host immune system and the growing tumor cells. This reactive space surrounding the tumor possesses several possibilities and facilitates the progression of a tumor from a neoplastic stage to that of metastasis. The contemporary approach of understanding the cancer biology from a "within the cell" perspective has been largely challenged with complex and intricate "outside the cell" events.

Clusterin protects neurons against intracellular proteotoxicity.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems.

MicroRNAs in HPV associated cancers: small players with big consequences.

MicroRNAs (miRs) are short (~20 nucleotides) non-coding ribonuecleic acids (ncRNAs) known to be involved in cellular processes such as proliferation, differentiation, immune response, pathogenicity and tumourigenesis, among many others. The regulatory mechanisms exerted by miRs have been implicated in many cancers, including Human Papillomavirus (HPV)-associated cancers. Areas covered: In this review, the authors discuss the involvement of miRs (-143, -375, -21, -200, -296 etc.

RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming.

Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process.