Beta-sheet Structure Research Articles

Unraveling the Membrane Topology of TMEM151A: A Step Towards Understanding its Cellular Role

Transmembrane protein 151A (TMEM151A) has been identified as a causative gene for paroxysmal kinesigenic dyskinesia, though its molecular function remains almost completely unknown. Understanding the membrane topology of transmembrane proteins is crucial for elucidating their functions and possible interacting partners. In this study, we utilized molecular dynamics simulations, immunocytochemistry, and electron microscopy to define the topology of TMEM151A. Our results validate a starting AlphaFold model of TMEM151A and reveal that it comprises a transmembrane domain with two membrane-spanning alpha helices connected by a short extracellular loop and an intramembrane helix-hinge-helix structure. Notably, most of the protein is oriented towards the intracellular side of the membranes with a large cytosolic domain featuring a combination of alpha-helix and beta-sheet structures, as well as the protein N- and C-termini. These insights into TMEM151A’s topology and orientation of its domains with respect of the cell membranes provide essential information for future functional studies and represent a first fundamental step for understanding its role in the pathogenesis of paroxysmal kinesigenic dyskinesia.

Designing novel multiepitope mRNA vaccine targeting Hendra virus (HeV): An integrative approach utilizing immunoinformatics, reverse vaccinology, and molecular dynamics simulation.

Human and animal health is threatened by Hendra virus (HeV), which has few treatments. This in-silico vaccine design study focuses on HeV G (glycoprotein), F (fusion protein), and M (matrix protein). These proteins were computationally assessed for B and T-cell epitopes after considering HeV strain conservation, immunogenicity, and antigenicity. To improve vaccination immunogenicity, these epitopes were selectively ligated into a multiepitope construct. To improve vaccination longevity and immunological response, adjuvants and linkers were ligated. G, F, and M epitopes were used to create an mRNA HeV vaccine. Cytotoxic, helper, and linear B-lymphocytes' epitopes are targeted by this vaccine. The population coverage analysis demonstrates that multi-epitope vaccination covers 91.81 percent of CTL and 98.55 percent of HTL epitopes worldwide. GRAVY evaluated the vaccine's well-characterized physicochemical properties -0.503, indicating solubility and functional stability. Structure analysis showed well-stabilized 2° and 3° structures in the vaccine, with alpha helix, beta sheet, and coil structures (Ramachandran score of 88.5% and Z score of -3.44). There was a strong affinity as shown by docking tests with TLR-4 (central score of -1139.4 KJ/mol) and TLR-2 (center score of -1277.9 KJ/mol). The coupled V-apo, V-TLR2, and V-TLR4 complexes were tested for binding using molecular dynamics simulation where extremely stable complexes were found. The predicted mRNA structures provided significant stability. Codon optimization for Escherichia. coli synthesis allowed the vaccine to attain a GC content of 46.83% and a CAI score of 1.0, which supports its significant expression. Immunological simulations indicated vaccine-induced innate and adaptive immune reactions. Finally, this potential HeV vaccine needs more studies to prove its efficacy and safety.

Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis.

This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2-14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4-8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens.

Tardigrade secretory proteins protect biological structures from desiccation

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focus on the proteins belonging to the secreted family of tardigrade proteins termed secretory-abundant heat soluble (“SAHS”) proteins, and investigate their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevent desiccated liposomes from fusion, and enhance desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

Rational design of structure-based vaccines targeting misfolded alpha-synuclein conformers of Parkinson's disease and related disorders.

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.

A solid beta-sheet structure is formed at the surface of FUS droplets during aging

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of β-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

Hidden imaging in thin polymer films with embedded fluorescent peptide nanodots.

Fluorescent (FL) encrypting nanostructures, such as quantum dots, carbon dots, organic dyes, lanthanide nanocrystals, DNA, and more, are effective tools for advanced applications in high-resolution hidden imaging. These applications include tracking, labeling, security printing, and anti-counterfeiting drug technology. In this work, what we believe to be a new FL encoding nanostructures has been proposed, which consists of recently discovered nanometer-scale peptide dots. When refolded into a beta-sheet peptide secondary structure, these biocompatible nanoparticles exhibit a strong and tunable FL effect. The biophotonic FL covers the entire visible spectrum, making the peptide dots next-generation nanoscale light sources with a quantum yield of 30%. Our studies demonstrate that these FL bio-nanodots also exhibit a significant irreversible photo-bleaching effect associated with the light-induced destruction of noncovalent intermolecular hydrogen bonds of the peptide dots' highly stable beta-sheet secondary structure. We present what we believe is a new approach for achieving high-resolution long-term optical memory by tailoring various hidden images in the developed thin polyvinyl alcohol (PVA) polymer films with an embedded dense array of FL peptide nanodots. The technology enables recording photo-bleached patterns, barcodes, and high-resolution images.

Disordered ganglioside localization in the Alzheimer’s disease brain

AbstractBackgroundAlzheimer’s disease (AD) is a progressive dementia that impairs memory and cognition. Gangliosides are glycosphingolipids enriched within the central nervous system and known to alter the secondary structure of amyloid beta deposits, a hallmark of AD (Choo‐Smith et al., 1997). The ganglioside GM1 is known to exert neuroprotective properties and was proposed as a therapeutic for AD (Svennerholm, 1994). In support of this, intraventricular administration of GM1 decreased amyloid beta deposits and provided clinical benefit (Augustinsson et al., 1997). Pre‐clinical work has since demonstrated a shift from complex GM1 species to simple GM2 and GM3 is linked to neurodegenerative pathogenesis (Wang & Whitehead, 2020). However, the ganglioside GM1 also converts amyloid beta to a toxic form with beta sheet structure (Yamamoto et al., 2007), and acts as a seed for amyloid beta fibrillogenesis (Yanagisawa et al., 1995). It is possible that different GM1 subtypes may be responsible for varying affects. Understanding the profile of GM1 subtypes in the AD brain may be a key step in capitalizing on its therapeutic potential. We investigate the ratio between 18 and 20 carbon subtypes of GM1 and simple ganglioside species in post‐mortem human AD brain tissue and non‐AD controls. Brain regions interrogated include the entorhinal cortex, basal forebrain, anterior commissure and hippocampus.MethodNeuroanatomical regions of interest (ROIs) were blocked from neuropathologically confirmed AD and non‐AD brains and stored in 10% formalin. Samples were prepared for matrix assisted laser desorption ionization imagining mass spectrometry (MALDI‐IMS) (Caughlin et al., 2017). MALDI‐IMS was performed in reflectron negative mode. Matching ROIs in AD and non‐AD samples (n = 5) were selected using TissueView software. Mass spectra were normalized by area under curve and peaks corresponding to gangliosides were statistically analyzed.ResultNon‐AD brains demonstrated distinct d20:1/d18:1 ratios of both simple and complex gangliosides between different brain regions. This expression profile was lost in the AD brain samples. Additionally, a significant decrease in this ratio was observed in AD compared to non‐AD samples in several brain regions.ConclusionGanglioside dysregulation and a decrease in the d20:1/d18:1 ratios in multiple brain regions are critical pathological biomarkers of AD.

Fate of pulse globulin proteins molecular Structure and composition on high moisture extrusion

Mungbean and lentil proteins are gaining attention for the production of high moisture extrusion (HME)-based meat- and dairy-analogues. However, understanding the mechanism of fibrous structure formation and molecular and higher-order changes in HME remains limited. Globulin protein isolates from mungbean (MBPI), green lentil (GLPI) and yellow pea (YPPI) along with commercial soy (CSPI) and yellow pea (CPPI) protein were investigated for their pasting properties using high-pressure rapid visco analyser (RVA), and the formation of anisotropic structures and molecular changes at constant HME conditions. Vicilin-rich MBPI showed higher viscosity on RVA, consumed higher specific mechanical energy (SME) during extrusion, and developed extrudates with higher textural strength and cutting force than proteins with both legumin and vicilin-like globulins, i.e. GLPI and YPPI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography (SEC) of extrudates' soluble proteins showed dissociation of vicilin-like globulins into their smaller subunits. In contrast, legumin-like globulins were reaggregated into higher molecular weight soluble macromolecules. Further, SDS-PAGE of extrudates’ total protein revealed the involvement of legumin-like globulin in the macrostructure network of the extrudates. During HME, native unfolded structures were destroyed with conversion, predominately driving changes in the tertiary structure (thermal denaturation) and secondary structure with loss of helical structure to formation of random coil and beta-sheet structures. Protein extractability in different solvents was greatly reduced, with covalent and non-covalent interactions being majorly involved in the stabilisation of extrudates structure. Protein composition, and in particular vicilin-legumin ratio and protein conformation, determined the structural development and molecular changes in HME.

Formation of amyloid fibrils from ovalbumin under Ohmic heating

Ohmic heating (OH) is an alternative sustainable heating technology that has demonstrated its potential to modify protein structures and aggregates. Furthermore, certain protein aggregates, namely amyloid fibrils (AF), are associated with an enhanced protein functionality, such as gelation. This study evaluates how Ohmic heating (OH) influences the formation of AF structures from ovalbumin source under two electric field strength levels, 8.5 to 10.5 and 24.0–31.0 V/cm, respectively. Hence, AF aggregate formation was assessed over holding times ranging from 30 to 1200 sunder various environmental conditions (3.45 and 67.95 mM NaCl, 80, 85 and 90 °C, pH = 7). AF were formed under all conditions. SDS-PAGE revealed that OH had a higher tendency to preserve native ovalbumin molecules. Furthermore, Congo Red and Thioflavin T stainings indicated that OH reduces the amount of AF structures. This finding was supported by FTIR measurements, which showed OH samples to contain lower amounts of beta-sheets. Field flow fractioning revealed smaller-sized aggregates or aggregate clusters occurred after OH treatment. In contrast, prolonged holding time or higher treatment temperatures increased ThT fluorescence, beta-sheet structures and aggregate as well as cluster sizes. Ionic strength was found to dominate the effects of electric field strength under different environmental conditions.

Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

Bovine liver catalase turns into three conformational states after exposure to an anionic surfactant.

The mechanism by which anionic surfactants promote amyloid fibril is not well understood. Here, we investigated how sodium dodecyl sulfate (SDS), a negatively charged surfactant, affects the fibrillation of the partially unfolded random-coiled bovine liver catalase (BLC) at a pH of 2.0. We used several methods, including turbidity, RLS kinetics, intrinsic fluorescence, ThT fluorescence, far-UV CD, and TEM imaging, to evaluate the conformational changes of BLC in vitro in response to SDS treatment. BLC is a multimeric protein and well folded at physiological pH but forms a random coil structure at pH 2.0. Intrinsic fluorescence and far-UV CD data showed that below 0.1mM SDS, random coiled BLC turned into a native-like structure. BLC incubated with an SDS concentration ranging from 0.1 to 2.0mM led to the formation of aggregates. The ThT fluorescence intensity was enhanced in the aggregated BLC samples (0.1-2.0mM SDS), and cross beta-sheeted structure was detected by the far UV CD measurements. BLC adopts a complete alpha-helical structure upon interacting with SDS at a more than 2.0mM concentration at pH 2.0. Understanding the mechanism of surfactant- or lipid-induced fibrillation is important for therapeutic purposes.

Non-Perturbative Identification and Subtyping of Amyloidosis in Human Kidney Tissue with Raman Spectroscopy and Machine Learning.

Amyloids are proteins with characteristic beta-sheet secondary structures that display fibrillary ultrastructural configurations. They can result in pathologic lesions when deposited in human organs. Various types of amyloid protein can be routinely identified in human tissue specimens by special stains, immunolabeling, and electron microscopy, and, for certain forms of amyloidosis, mass spectrometry is required. In this study, we applied Raman spectroscopy to identify immunoglobulin light chain and amyloid A amyloidosis in human renal tissue biopsies and compared the results with a normal kidney biopsy as a control case. Raman spectra of amyloid fibrils within unstained, frozen, human kidney tissue demonstrated changes in conformation of protein secondary structures. By using t-distributed stochastic neighbor embedding (t-SNE) and density-based spatial clustering of applications with noise (DBSCAN), Raman spectroscopic data were accurately classified with respect to each amyloid type and deposition site. To the best of our knowledge, this is the first time Raman spectroscopy has been used for amyloid characterization of ex vivo human kidney tissue samples. Our approach, using Raman spectroscopy with machine learning algorithms, shows the potential for the identification of amyloid in pathologic lesions.

Ohmic vs. conventional heating: Influence of moderate electric fields on properties of potato protein isolate gels

The influence of moderate electric fields (MEF) on thermally induced gelation and network structures of patatin enriched potato protein (PPI) was investigated. PPI solutions with 9 wt% protein (pH 7) and 25 mM NaCl were heated from 25 to 65 °C via OH (3–24 V/cm) or conventional heating (COV) at various come-up (240 s and 1200 s) and holding times (30 s and 600 s). Self-standing gels were produced but less proteins denatured when heated via OH. Further, SDS-PAGE and GPC measurements revealed more native patatin remaining after OH treatment. Scanning electron microscopy showed OH gels to have more gap-like structures and frayed areas than COV treated gels which resulted in lower water holding capacity. On molecular scale, less hydrophobic interactions were measured within the protein network and FTIR trials showed the MEF to affect beta-sheet structures. OH gels further showed lower rigidity and higher flexibility, thus, gelling functionality was affected via OH.Industrial relevance: Ohmic heating (OH) is an advanced heating technology which has gained interest within the last decades as it is a highly energy and time efficient way to heat up foods. Additionally, as OH does not require any fossil energies, OH is considered to be a sustainable heating option for the food processing industry. During OH, electrical current is converted into heat within the food. However, it is not fully understood yet, whether OH influences the food matrix compared to conventional heating and how OH impacts certain ingredients, e.g. globular proteins and their denaturation/ gelation. This study helps to provide knowledge facilitating an implementation of OH into industry processes, serving a more sustainable food production. To evaluate the influence of OH on protein gelation, a patatin enriched potato protein was chosen as this protein offers a high protein functionality and therefore is of high interest for food industry (e.g. to be used as ingredient for plant based foods).

Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aβ) peptides

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.

Evaluation of effects of temperature and humidity on the secondary structure of Ganirelix in an injectable formulation and comparison with Orgalutran® using circular dichroism spectroscopy.

Ganirelix, a drug used in in vitro fertilization (IVF), prevents ovulation in women who are not ready to have children by inhibiting a gene that produces gonadotropin. Peptides are macromolecules that are able to preserve a predetermined shape while carrying out the structural and regulatory roles for which they were originally intended. Peptide structures can be altered in the production and storage processes. Therapeutic peptides' biological activity can be drastically altered by even small modifications in their primary and secondary structures. The molecules' secondary structures can be monitored by subjecting them to different processing or storage conditions. In our investigation, we used circular dichroism (CD) spectroscopy with two different software programs for secondary structure evaluation to look at how environmental factors like temperature and humidity affected the secondary structure of Ganirelix in an injectable formulation. The CD results revealed that the alpha-helical (regular and distorted), beta-sheet, beta-strands (regular and distorted), beta-turn, and random coil structures of temperature and humidity stressed generic drug products are comparable to reference-listed drug.

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

SARS-CoV-2 Orf1ab Genome Mutations, the Driving Force for Virus Pathogenicity.

Background: The proteins codified in the Open Reading Frame 1ab -Orf1ab- region of the SARS-CoV-2 genome are the main responsible of the virus transcription, replication, and translation processes inside the human cell. Once inside the cell, the viral mRNA encode structural and nonstructural proteins, that direct virus assembly, transcription, replication and host control, and the accessory proteins whose function has not been determined. The largest gene, Orf1ab, contains overlapping open reading frames that encode polyproteins PP1ab and PP1a. These polyproteins are cleaved to yield 16 nonstructural proteins, NSP1-16. Production of the longer (PP1ab) or shorter protein (PP1a) depends on a ribosomal frameshifting event. The proteins, include the papain-like proteinase (NSP3), 3C-like proteinase (NSP5), RNA-dependent RNA polymerase (NSP12, RdRp), helicase (NSP13, HEL), endoRNAse (NSP15), 2'-O-Ribose-Methyltransferase (NSP16) and other nonstructural proteins. The SARS-CoV-2 nonstructural proteins are responsible for viral transcription, replication, proteolytic processing, suppression of host immune responses, and suppression of host gene expression. The purpose of the manuscript is to present a systematic review as of September 30, 2023, on the Orf1ab region mutations of the SARS-CoV-2 genome as of September 30, 2023, with the aim to predict, through the mutations profile on that region, the severity of an infection for a new SARS-CoV-2 variant that could emerge in the near future. Material and Methods: Original scientific articles published in Medline, Pubmed, Science Direct, Web of Science, Scopus, EBSCO and BioMed Central databases, official health organizations (WHO, CDC, ECDEC, NIH) electronic publications, and specialized media in the subject, were electronically searched to accomplish the aim of the study. Articles published in any language were included from 2020 to present using a variety of keywords in combination. The studies relevant to our review were analysed and compared. Results and discussion: The NIH “National Human Genome Research Institute” define the Open Reading Frames (ORFs) as a portion of a DNA sequence that does not include a stop codon. The Open Reading Frames encode accessory proteins transcribed from the 3′ one-third of the genome to form a set of subgenomic mRNAs (sg mRNAs). Following entry on the human cell, the viral particle release the genomic RNA molecule that is translated on two large open reading frames, ORF1a and ORF1b. The resulting polyproteins pp1a and pp1ab are co-translationally and post-translationally processed into the individual non-structural proteins (NSPs) that form the viral replication and transcription complex (RTC). Translated structural proteins translocate into endoplasmic reticulum (ER) membranes and transit through the ER-to-Golgi intermediate compartment (ERGIC), where interaction with N-encapsidated, newly produced genomic RNA results in budding into the lumen of secretory vesicular compartments. Two viral proteases, Plpro -NSP3- and 3Cl-pro -NSP5-, process the polyproteins and generate the nonstructural proteins NSP1-NSP16 that directs the transcription, replication and construction of new virions that are secreted by exocytosis from the infected cell. The evolution of key proteins in viral transcription and replication is clearly observed by carefully studying the structure, function, and evolution of RdRp, Mpro or 3Clpro, and NSP13 proteins directed by the Orf1a and Orf1ab genome mutations. Conclusions: ORF1ab is cleaved into 16 non-structural proteins involved in SARS-CoV-2 transcription and genome replication. P323L, P227L, G671S, V776L and A185S are the first five frequent mutations of RdRp (NSP12), the mutations P227L and G671S might have functional consequences in the viral transcription and replication. Mutations in residues D499 to L514, K545, R555, T611 to M626, G678 to T710, S759 to D761 are directly implicated with the transcription-replication capability of the virus by RdRp. In Mpro (NSP5) the mutation of residues H41, P132, C145, S145, L226, T234, R298, S301, F305, and Q306 may increase the efficiency of proteolytic cleavage of proteins such as NEMO, thereby improving the ability of the omicron series of viruses to suppress the immune system and accelerate the viral replication. In Helicase (NSP13) the mutations of residues E261, K218, K288, S289, H290, D374, E375, Q404, K460, R567, and A598 are involved in the separation of the double-stranded RNA or DNA with a 5′→3′ polarity as well as 5′ mRNA capping activity in the virus transcription-replication process. In the Orf1ab gene, ORF1b:V2354F mutation, corresponding to NSP15:V303F, may induce a conformational change and result in a disruption to a flanking beta-sheet structure. The premature stop codon ORF7a:Q94*, truncates the transmembrane protein and cytosolic tail used to mediate protein transport, may affect protein localization to the ER-Golgi. The analyses of Orf1ab genome mutations, allows us to predict, through the mutations profile on that region, the severity of an infection for a new SARS-CoV-2 variant that could emerge in the near future.

Application of PEG-modified Copper-cysteamine in photodynamic therapy for Alzheimer's disease

• The Cu-Cy and Cu-Cy-PEG were synthetized, and the biosafety of Cu-Cy-PEG was improved. • Cu-Cy-PEG can generate ROS efficiently and be used to degrade Aβ aggregates. • The beta-sheet secondary structure of amyloid fibrils was destroyed in PDT. As a progressive neurodegenerative disease, Alzheimer's disease (AD) is characterized by the accumulation and misfolding of amyloid polypeptides (Aβ) which results in irreversible brain damages, cognitive decline and behavioral impairment. Photodynamic therapy (PDT) has received much attention as a potential approach for AD treatment. In this study, we investigated a novel multifunctional photosensitizer, copper-cysteamine (Cu-Cy). With PEG modification on the surface (Cu-Cy-PEG), the particle size and cytotoxicity of Cu-Cy-PEG were significantly reduced compared with Cu-Cy. Under UV irradiation, Cu-Cy-PEG can produce reactive oxygen species (ROS) and could degrade Aβ(1-40) aggregates efficiently. The macroscopic amyloid aggregates were photodegraded by Cu-Cy-PEG into amorphous particles. This is the first case for Cu-Cy-PEG being used for degrading Aβ aggregates in vitro, which proved that Cu-Cy-PEG may serve as a potential treatment in AD therapy.

A Computer Simulation Insight into the Formation of Apocarotenoids: Study of the Carotenoid Oxygenases BCO1 and BCO2 and Their Interaction with Putative Substrates.

Carotenoids are isoprenoid pigments, and sources of vitamin A in humans. The first metabolic pathway for their synthesis is mediated by the enzymes β,β-carotene-15,15'-dioxygenase (BCO1) and β,β-carotene-9',10'-dioxygenase (BCO2), which cleave carotenoids into smaller compounds, called apocarotenoids. The objective of this study is to gain insight into the interaction of BCO1 and BCO2 with carotenoids, adding structural diversity and importance in the agro-food and/or health sectors. Homology modeling of BCO1 and BCO2, and the molecular dynamics of complexes with all carotenoids were performed. Interaction energy and structures were analyzed. For both enzymes, the general structure is conserved with a seven beta-sheet structure, and the β-carotene is positioned at an optimal distance from the catalytic center. Fe2+ forms in an octahedral coordination sphere with four perfectly conserved histidine residues. BCO1 finds stability in a structure in which the β-carotene is positioned ready for enzymatic catalysis at the 15-15' bond, and BCO2 in positioning the bond to be cleaved (C9-C10) close to the active site. In BCO1 the carotenoids interact with only seven residues with aromatic rings, while the interaction of BCO2 is much more varied in terms of the type of interaction, with more residues of different chemical natures.