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The Clinical Importance of Botulinum Toxin as an Injected Protein
Published in Yates Yen-Yu Chao, Optimizing Aesthetic Toxin Results, 2022
BoNT/A can denature during the process of the production, purification, and pharmaceutical preparation of the drug, and during the reconstitution of solid formulations before use. The reasons for this can be exposure to heat, digestion by proteases, severe agitation, and very high salt concentrations during the preparation of the solid product. Denatured proteins tend to form protein aggregates. DCs recognize protein aggregates and are strongly activated. There is no dearth of literature on the immunogenicity of protein aggregates in biologicals (e.g. Rosenberg 2006).
Order Sepolyvirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
In insect cells, the efficient production of the virion-like JCPyV VP1 VLPs was demonstrated (Chang D et al. 1997; Goldmann et al. 1999; Viscidi et al. 2011; Šroller et al. 2014). In E. coli, Ou et al. (1999) demonstrated that the JCPyV VP1 self-assembled into the VLPs represented by the virion-like pseudovirion and empty capsid-like populations. The pseudovirions contained DNA and RNA molecules, but the pseudocapsids did not contain any nucleic acid. Moreover, the pseudovirions were able to package and deliver exogenous DNA into human fetal kidney epithelial cells, acting as a potent human gene transfer vector (Ou et al. 1999). To investigate the minimal sequences on the JCPyV VP1 required for the assembly, Ou et al. (2001) truncated it by the first 12 and 19 aa at the N-terminus and the last 16, 17, and 31 aa at the C-terminus. The ΔN12 and ΔC16 VP1 variants were self-assembly competent, while the ΔN19, ΔC17, and ΔC31 formed a pentameric capsomere structure only. It should be noted that the E. coli chaperone DnaK is incredibly important for the correct VP1 assembly and reduction of protein aggregates (Saccardo et al. 2014).
Approach to Vacuolar Myopathy
Published in Maher Kurdi, Neuromuscular Pathology Made Easy, 2021
It has been scientifically proven that autophagic dysfunction during the cellular catabolic process is considered the main predisposing factor for vacuolar myopathy. This impairment prevents the elimination of misfolded protein aggregates and increases the ability of cellular oxidative stress. As a result, lysosomal breakdown occurs. Several mechanisms in the literature have explained the pathogenesis of these vacuoles (Figure 15.1). A selective impairment of cargo sequestration is often encountered in vacuolar myopathies. This may cause a defected chaperone-assisted selective autophagy (CASA) that forms protein-containing vacuoles such as myofibrillar myopathies (MFMs).
Novel therapeutic targets for amyotrophic lateral sclerosis: ribonucleoproteins and cellular autonomy
Published in Expert Opinion on Therapeutic Targets, 2020
RBP mislocalization from the nucleus to the cytoplasm is also associated with misfolding and cytoplasmic aggregation in ALS. Initiating events of this process possibly relate to deregulated liquid-liquid phase separation (LLPS). From the viewpoint of RBP aggregation, the ALS field has benefited from studies in the prototypic protein misfolding disorders, the prion diseases. It is widely accepted that misfolded proteins underlie the cellular pathogenesis and cell-to-cell propagation in classic prion diseases by forming distinct conformations of amyloid cross β sheets, which then serve as self-templates recruiting native monomers to misfold. Indeed, analogous ‘seeding’/propagation phenomena have been demonstrated experimentally for different ALS proteins including TDP-43 [56,57], FUS [58] and SOD1 [59]. These experimental models usually rely on protein overexpression in non-human or non-neuronal cell lines. However, we have recently demonstrated seeded aggregation in hiPSC-derived motor neurons treated with serially passaged sarkosyl-insoluble extract from sporadic ALS postmortem tissue. In this work, we also demonstrated that TDP-43 oligomers are at least part of the toxic principle in ALS [60]. This raises the possibility of designing therapeutics that target these toxic oligomers. Broadly, three main strategies can be considered here: i) perturbing the formation of protein aggregates within the cell; ii) promoting their clearance from affected cells and iii) preventing their uptake into other cells. These approaches are discussed in more detail below.
Methods for detecting toxic α-synuclein species as a biomarker for Parkinson’s disease
Published in Critical Reviews in Clinical Laboratory Sciences, 2020
Darren M. O’Hara, Suneil K. Kalia, Lorraine V. Kalia
PD is classified as an synucleinopathy, part of a wider group of diseases characterized by intracellular aggregation of the protein α-synuclein (α-syn). Other synucleinopathies include dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). In PD and DLB, the defining protein aggregate is an accumulation of insoluble, filamentous inclusions known as Lewy bodies (LB) and Lewy neurites, which are composed primarily of α-syn [3]. A small proportion of PD cases (∼10%) arise as a result of mutations in a specific subset of genes, including SNCA (which encodes α-syn), PRKN (which encodes parkin), PINK1, and LRRK2 [4]. Histopathologically, comparisons of postmortem brains of patients with genetic and non-genetic forms of PD show similar patterns of α-syn deposition in LB, except for parkin-, PINK1-, and LRRK2-related PD in which LB are sometimes absent [5,6]. Proteins encoded by PD-related genes are involved in many cellular processes, including mitochondrial quality control and protein degradation. Yet, despite extensive research, little is known about the specific cause of dopaminergic cell death within the SN of PD patients.
Defining the right diluent for intravenous infusion of therapeutic antibodies
Published in mAbs, 2020
Shen Luo, Keisha Melodi McSweeney, Tao Wang, Silvia M. Bacot, Gerald M. Feldman, Baolin Zhang
To identify protein composition, insoluble protein aggregates were subjected to in-solution or in-gel digestion followed by LC-MS/MS analysis. For in-solution digestion, protein pellets were dissolved in 100 µL of 6 M guanidine hydrochloride in 100 mM Tris, 1 mM EDTA, pH 8.5. Reduction and alkylation of cysteine residues were achieved by adding 1 µL 0.5 M DTT and incubation at 37°C for 30 minutes followed by adding 6 µL 375 mM iodoacetamide (ThermoFisher, cat. no. A39271) and incubating for an additional 30 minutes in dark. After adding 2 µL 0.5 M DTT and incubation at room temperature for 5 minutes to terminate the reaction, 110 µL ice cold 20% trichloroacetic acid was mixed with the solution on ice for 5 minutes to allow precipitation of both proteins and guanidine. After centrifugation at 4°C and 21,000 g for 3 minutes, guanidine in the resultant pellets was removed by washing three times with an ice-cold 0.5 mL 1:1 (v/v) mixture of ethanol and ethyl acetate. The protein pellets were air dried and dissolved in 100 µL 25 mM ammonium bicarbonate. Tryptic protein digestion was initiated by adding 0.5 µg trypsin and incubated at 37°C overnight; the digestion was stopped by adding 5 µL 10% formic acid. The solution was transferred into an HPLC vial. The tryptic peptides were dried in a Savant SpeedVac concentrator (ThermoFisher) and dissolved in 10 µL 3% acetonitrile in 0.1% formic acid.