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Biomolecules Jun 2024Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead...
Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is the presence of one additional oxygen atom. To elucidate why such a subtle difference in ligand structure can result in different typical or atypical profiles, we investigated two pairs of analogs. Our quantum mechanical calculations revealed a more negatively charged distribution of the electrostatic potential surface of the sulfoxide substitution. Using molecular dynamics simulations, we demonstrated that sulfoxide-substituted modafinil analogs have a propensity to attract more water into the binding pocket. They also exhibited a tendency to dissociate from Asp79 and form a new interaction with Asp421, consequently promoting an inward-facing conformation of hDAT. In contrast, sulfide-substituted analogs did not display these effects. These findings elucidate the structural basis of the activity cliff observed with modafinil analogs and also enhance our understanding of the functionally relevant conformational spectrum of hDAT.
Topics: Modafinil; Dopamine Plasma Membrane Transport Proteins; Humans; Molecular Dynamics Simulation; Binding Sites; Dopamine Uptake Inhibitors; Structure-Activity Relationship; Protein Binding
PubMed: 38927116
DOI: 10.3390/biom14060713 -
Biomolecules Jun 2024Reactive oxygen species (ROS) contain at least one oxygen atom and one or more unpaired electrons and include singlet oxygen, superoxide anion radical, hydroxyl radical,... (Review)
Review
Reactive oxygen species (ROS) contain at least one oxygen atom and one or more unpaired electrons and include singlet oxygen, superoxide anion radical, hydroxyl radical, hydroperoxyl radical, and free nitrogen radicals. Intracellular ROS can be formed as a consequence of several factors, including ultra-violet (UV) radiation, electron leakage during aerobic respiration, inflammatory responses mediated by macrophages, and other external stimuli or stress. The enhanced production of ROS is termed oxidative stress and this leads to cellular damage, such as protein carbonylation, lipid peroxidation, deoxyribonucleic acid (DNA) damage, and base modifications. This damage may manifest in various pathological states, including ageing, cancer, neurological diseases, and metabolic disorders like diabetes. On the other hand, the optimum levels of ROS have been implicated in the regulation of many important physiological processes. For example, the ROS generated in the mitochondria (mitochondrial ROS or mt-ROS), as a byproduct of the electron transport chain (ETC), participate in a plethora of physiological functions, which include ageing, cell growth, cell proliferation, and immune response and regulation. In this current review, we will focus on the mechanisms by which mt-ROS regulate different pathways of host immune responses in the context of infection by bacteria, protozoan parasites, viruses, and fungi. We will also discuss how these pathogens, in turn, modulate mt-ROS to evade host immunity. We will conclude by briefly giving an overview of the potential therapeutic approaches involving mt-ROS in infectious diseases.
Topics: Reactive Oxygen Species; Humans; Mitochondria; Animals; Oxidative Stress; Infections; Immunity
PubMed: 38927073
DOI: 10.3390/biom14060670 -
Biomolecules Jun 2024Abdominal aortic aneurysm (AAA) is a progressive dilatation of the aorta that can lead to aortic rupture. The pathophysiology of the disease is not well characterized... (Review)
Review
Abdominal aortic aneurysm (AAA) is a progressive dilatation of the aorta that can lead to aortic rupture. The pathophysiology of the disease is not well characterized but is known to be caused by the general breakdown of the extracellular matrix within the aortic wall. In this comprehensive literature review, all current research on proteins that have been investigated for their potential prognostic capabilities in patients with AAA was included. A total of 45 proteins were found to be potential prognostic biomarkers for AAA, predicting incidence of AAA, AAA rupture, AAA growth, endoleak, and post-surgical mortality. The 45 proteins fell into the following seven general categories based on their primary function: (1) cardiovascular health, (2) hemostasis, (3) transport proteins, (4) inflammation and immunity, (5) kidney function, (6) cellular structure, (7) and hormones and growth factors. This is the most up-to-date literature review on current prognostic markers for AAA and their functions. This review outlines the wide pathophysiological processes that are implicated in AAA disease progression.
Topics: Aortic Aneurysm, Abdominal; Humans; Biomarkers; Prognosis
PubMed: 38927064
DOI: 10.3390/biom14060661 -
Modulation of Adverse Health Effects of Environmental Cadmium Exposure by Zinc and Its Transporters.Biomolecules May 2024Zinc (Zn) is the second most abundant metal in the human body and is essential for the function of 10% of all proteins. As metals cannot be synthesized or degraded, they... (Review)
Review
Zinc (Zn) is the second most abundant metal in the human body and is essential for the function of 10% of all proteins. As metals cannot be synthesized or degraded, they must be assimilated from the diet by specialized transport proteins, which unfortunately also provide an entry route for the toxic metal pollutant cadmium (Cd). The intestinal absorption of Zn depends on the composition of food that is consumed, firstly the amount of Zn itself and then the quantity of other food constituents such as phytate, protein, and calcium (Ca). In cells, Zn is involved in the regulation of intermediary metabolism, gene expression, cell growth, differentiation, apoptosis, and antioxidant defense mechanisms. The cellular influx, efflux, subcellular compartmentalization, and trafficking of Zn are coordinated by transporter proteins, solute-linked carriers 30A and 39A (SLC30A and SLC39A), known as the ZnT and Zrt/Irt-like protein (ZIP). Because of its chemical similarity with Zn and Ca, Cd disrupts the physiological functions of both. The concurrent induction of a Zn efflux transporter ZnT1 (SLC30A1) and metallothionein by Cd disrupts the homeostasis and reduces the bioavailability of Zn. The present review highlights the increased mortality and the severity of various diseases among Cd-exposed persons and the roles of Zn and other transport proteins in the manifestation of Cd cytotoxicity. Special emphasis is given to Zn intake levels that may lower the risk of vision loss and bone fracture associated with Cd exposure. The difficult challenge of determining a permissible intake level of Cd is discussed in relation to the recommended dietary Zn intake levels.
Topics: Humans; Cadmium; Zinc; Environmental Exposure; Animals; Cation Transport Proteins; Metallothionein
PubMed: 38927054
DOI: 10.3390/biom14060650 -
MSphere Jun 2024The genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly...
The genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops , we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of and commensal species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for infection and immune avoidance.
PubMed: 38926904
DOI: 10.1128/msphere.00450-24 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
BMC Biology Jun 2024The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans....
BACKGROUND
The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown.
RESULTS
To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments.
CONCLUSIONS
We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.
Topics: Animals; Mice; Mice, Knockout; Synaptic Vesicles; Vacuolar Proton-Translocating ATPases; Synaptic Transmission; Brain; Behavior, Animal; Synapses; Neurons; Vesicular Transport Proteins
PubMed: 38926759
DOI: 10.1186/s12915-024-01940-y -
Communications Biology Jun 2024In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses...
In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses the flux through the PstSCAB phosphate transporter as a readout of the extracellular phosphate level to control phosphate-responsive genes. While the sensing of environmental phosphate is well-investigated, the regulatory effects of cytoplasmic phosphate are unclear. Here, we disentangle the physiological and transcriptional responses of Caulobacter crescentus to changes in the environmental and cytoplasmic phosphate levels by uncoupling phosphate uptake from the activity of the PstSCAB system, using an additional, heterologously produced phosphate transporter. This approach reveals a two-pronged response of C. crescentus to phosphate limitation, in which PhoR-PhoB signaling mostly facilitates the utilization of alternative phosphate sources, whereas the cytoplasmic phosphate level controls the morphological and physiological adaptation of cells to growth under global phosphate limitation. These findings open the door to a comprehensive understanding of phosphate signaling in bacteria.
Topics: Caulobacter crescentus; Phosphates; Bacterial Proteins; Gene Expression Regulation, Bacterial; Cytoplasm; Signal Transduction; Phosphate Transport Proteins
PubMed: 38926609
DOI: 10.1038/s42003-024-06469-y -
Scientific Reports Jun 2024To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA...
To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA and protein levels via the use of recombinant carriers in vitro. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the pathogenic genes of the child and validate the mutations in the parents. pEGFP and phage vectors carrying wild-type and mutant OCA2 were constructed using the coding DNA sequence (CDS) of the whole gene-synthesized OCA2 as a template and transfected into HEK293T cells, after which expression analysis was performed. The child in this study was born with white skin, hair, eyelashes, and eyebrows and exhibited nystagmus. Genetic analysis indicated that the child carried two heterozygous mutations: c.1079C > T (p.Ser360Phe) of maternal origin and c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) of paternal origin, conforming to an autosomal recessive inheritance pattern. In vitro analysis showed that the expression of the c.1079C > T (p.Ser360Phe) mutant did not significantly change at the mRNA level but did increase at the protein level, suggesting that the mutation may lead to enhanced protein stability, and the c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) mutation resulted in the loss of three amino acids in exon 10, producing a truncated protein. In vitro expression analysis also revealed that the expression of the mutant gene was significantly downregulated at both the mRNA and protein levels, suggesting that the mutation can simultaneously produce truncated proteins and lead to protein degradation. This case study enriches the phenotypic spectrum of OCA2 gene disease. In vitro expression analysis confirmed that both mutations affect protein expression, providing a theoretical basis for analysing the pathogenicity of these two mutations.
Topics: Humans; HEK293 Cells; Mutation; Albinism, Oculocutaneous; Membrane Transport Proteins; Exome Sequencing; Female; Male; Pedigree; RNA, Messenger
PubMed: 38926510
DOI: 10.1038/s41598-024-64782-2 -
Life Science Alliance Sep 2024In , inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNAi...
In , inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNAi defective () loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of gene products-hSIDT1 and hSIDT2-mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules-a flexible lipid binding domain and a rigid transmembrane domain core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members.
Topics: Humans; Membrane Proteins; Protein Binding; Protein Domains; Models, Molecular; Protein Conformation; Caenorhabditis elegans; Animals; Amino Acid Sequence; Binding Sites; Lipids; Caenorhabditis elegans Proteins; RNA Interference
PubMed: 38925866
DOI: 10.26508/lsa.202402624