Search Results (161)

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae. Full article

The increasing incidence of cutaneous squamous cell carcinoma (cSCC), together with the ominous risks of metastasis and recurrence, underscores the importance of identifying novel therapies and validated biomarkers to augment patient management, particularly in the context of well-established and advanced disease. Following a brief overview of the well-recognized epidemiology, clinical features, and diagnosis of cSCC, the current review is focused on risk factors, most prominently excessive exposure to ultraviolet radiation (UVR) as a cause of persistent, pro-tumorigenic mutagenesis, and immune suppression. The next phase of the review encompasses an evaluation of the search for key driver mutations in the pathogenesis of cSCC, including the role of these and other mutations in the formation of immunologically reactive neoepitopes. With respect to additional mechanisms of tumorigenesis, immune evasion is prioritized, specifically the involvement of cell-free and infiltrating cellular mediators of immune suppression. Prominent amongst the former are the cytokine, transforming growth factor-β1 (TGF-β1), the prostanoid, prostaglandin E2, and the emerging immune suppressive nucleoside adenosine. In the case of the latter, tumor-infiltrating and circulating regulatory T cells have been implicated as being key players. The final sections of the review are focused on an update of the immunotherapy of established and advanced disease, as well as on the search for novel, reliable lesional and systemic biomarkers with the potential to guide patient management. Full article

In the mid-1970s, it was revealed that the 5′ end of the RNA genome of poliovirus (PV) was covalently linked to a peptide called VPg (viral protein, genome-linked). Subsequently, VPgs have been found attached to many other viruses and even phages. This review summarizes the patterns of physicochemical properties that are conserved within the VPgs of plus-strand RNA viruses where short-peptide VPgs have been identified. Mutagenesis and structural data indicate the importance of a 5 aa conserved motif at the N-termini of picornaviral VPgs (around the tyrosine 3 residue, which forms a covalent bond to UMP and the RNA). Hidden Markov models have been used to find motifs and VPgs in additional genera of picornaviruses, as well as dicistroviruses in insects and comoviruses in plants. These latter VPgs are bound to the RNA termina through linkages to serine or threonine. The role of free VPg and VPgpU needs clarification, especially in light of multiple genome copies in many of the viruses. Lysine and other positively charged side chains are hallmarks of VPgs. These may contribute to interactions with the viral RNA, polymerase, membranes and cellular proteins. The larger protein VPgs from potyviruses and noroviruses/caliciviruses may also show some areas of similar properties to these small peptides. Full article

Adenylosuccinate synthetase (AdSS), encoded by the ADE12 gene in yeast Saccharomyces cerevisiae, plays a critical role in purine biosynthesis, catalyzing the conversion of inosine 5′-monophosphate (IMP) and aspartic acid to adenylosuccinate, a substrate for the following adenosine 5′-monophosphate (AMP) synthesis step. Mutants lacking AdSS activity exhibit a range of pleiotropic phenotypes: slow growth, poor spore germination, accumulation, and secretion of inosine and hypoxanthine. We report new phenotypes of ade12 mutants and explain their molecular mechanisms. A GC-MS analysis showed that ade12 mutants have highly altered metabolite profiles: the accumulation of IMP leads to an impaired cellular energy metabolism, resulting in a dysregulation of key processes—the metabolism of nucleotides, carbohydrates, and amino acids. These metabolic perturbations explain the cell division arrest observed in ade12 yeast strains. A slowed replication in ade12 mutants, because of the insufficient availability of energy, nucleotides, and proteins, leads to the error-prone DNA polymerase ζ-dependent elevation of spontaneous mutagenesis, connecting multiple roles of AdSS in metabolism with genome stability control. Full article

DNA metabolism consists of crucial processes occurring in all living cells. These processes include various transactions, such as DNA replication, genetic recombination, transposition, mutagenesis, and DNA repair. While it was initially assumed that these processes might occur in the cytoplasm of prokaryotic cells, subsequent reports indicated the importance of the cell membrane in various DNA transactions. Furthermore, newly identified factors play significant roles in regulating DNA-related cellular processes. One such factor is the Hfq protein, originally discovered as an RNA chaperone but later shown to be involved in several molecular mechanisms. These include DNA transactions and interaction with the cell membrane. Recent studies have suggested that Hfq plays a role in the regulation of DNA replication, mutagenesis, and recombination. In this narrative review, we will focus on the importance of membranes in DNA transactions and discuss the potential role of Hfq-mediated regulation of these processes in Escherichia coli, where the protein is the best characterized. Special attention is given to the affinity of this small protein for both DNA and membranes, which might help explain some of the findings from recent experiments. Full article

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising the pore-forming subunit Ca_Vα₁ and the ancillary proteins Ca_Vα₂δ and Ca_Vβ. They are expressed in various tissues, including the nervous system, where they regulate Ca²⁺ entry in response to membrane potential changes. The increase in intracellular Ca²⁺ allows for regulating cell excitability and releasing neurotransmitters, among other cellular events. Leucine-rich repeat kinase 2 (LRRK2) is a serine–threonine kinase involved in vesicular mobilization. Previously, it has been shown that LRRK2 regulates neurotransmission by phosphorylating the Ca_Vβ auxiliary subunit of the Ca_V2.1 (P/Q-type) presynaptic channels. However, it is unknown whether the kinase can regulate the activity of other Ca_V channel subtypes, such as Ca_V1.3 (L-type), which play a significant role in the excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) and whose dysregulation contributes to neurodegeneration in Parkinson’s disease (PD). Here, we found potential phosphorylation sites for LRRK2 in Ca_Vβ₃ and examined how these molecules interact. We used immunoprecipitation and electrophysiology in HEK-293 cells expressing recombinant Ca_V1.3 channels, both with and without wild-type LRRK2 or its LRRK2^G2019S mutation, which plays a role in familial PD through a possible gain-of-toxic-function mechanism. Our results show that LRRK2^G2019S significantly increases current density through Ca_V1.3 channels, and this effect depends on the presence of Ca_Vβ₃. Site-directed mutagenesis revealed that phosphorylation at S152 in the sequence of Ca_Vβ₃ is necessary and sufficient to explain the abnormal regulation of the channels mediated by LRRK2^G2019S. These data provide new insights into the molecular regulation that mutant LRRK2 may exert on L-type Ca_V1.3 channels, which determine pacemaker activity in dopaminergic neurons of the SNc and may, therefore, play a relevant role in the molecular pathophysiology of PD. Full article

Human cysteine desulfurase (NFS1) participates in numerous critical cellular processes, including iron–sulfur (Fe-S) cluster biosynthesis and tRNA thiolation. NFS1 overexpression has been observed in a variety of cancers, and thus it has been considered a promising anti-tumor therapeutic target. To date, however, no inhibitors targeting NFS1 have been identified. Here, we report the identification of the first potent small-molecule inhibitor (Compound 53, PubChem CID 136847320) of NFS1 through a combination of virtual screening and biological validation. Compound 53 exhibited good selectivity against two other pyridoxal phosphate (PLP)-dependent enzymes. Treatment with Compound 53 inhibited the proliferation of lung cancer (A549) cells (IC₅₀ = 16.3 ± 1.92 μM) and caused an increase in cellular iron levels due to the disruption of Fe-S cluster biogenesis. Furthermore, Compound 53, in combination with 2-AAPA, an inhibitor of glutathione reductase (GR) that elevates cellular reactive oxygen species (ROS) levels, further suppressed the proliferation of A549 cells by triggering ferroptotic cell death. Additionally, the key residues involved in the binding of the inhibitor to the active center of NFS1 were identified through a combination of molecular docking and site-directed mutagenesis. Taken together, we describe the identification of the first selective small-molecule inhibitor of human NFS1. Full article

The genus Nannochloropsis comprises a group of oleaginous microalgae that accumulate polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA). These molecules are essential for the correct development and health of humans and animals. Thanks to their attractive lipid profile, Nannochloropsis is mainly marketed as a feed ingredient in aquaculture. In microalgae of this genus, contents and cellular location of PUFAs are affected by the growth conditions and gene expression. Strain improvement through non-recombinant approaches can generate more productive strains and efficient bioprocesses for PUFA production. Nevertheless, the lack of specific markers, detection methods, and selective pressure for isolating such mutants remains a bottleneck in classical mutagenesis approaches or lipid quality assessment during cultivation. This review encompasses the importance of PUFAs and lipid classes from Nannochloropsis species and their potential applications. Additionally, a revision of the different ways to increase PUFA content in Nannochloropsis sp. by using classical mutagenesis and adaptive laboratory evolution is also presented, as well as various methods to label and quantify lipids and PUFAs from Nannochloropsis microalgae. Full article

Background: Pancreas disease (PD) is a serious disease in European salmonid aquaculture caused by salmonid alphavirus (SAV), of which six genotypes (SAV1–6) have been described. The use of inactivated virus and DNA PD vaccines is common in marine salmonid aquaculture and has contributed to a reduction of the occurrence of disease; however, outbreaks are still frequent. Methods: In this study, we compared the long-term protection after immunization of Atlantic salmon (Salmo salar) with three different clones of attenuated infectious SAV3. The clones were made by site-directed mutagenesis targeting the glycoprotein E2 to disrupt the viral attachment and/or nuclear localization signal (NLS) of the capsid protein to disrupt the viral suppression of cellular nuclear-cytosol trafficking. The resulting clones (Clones 1–3) were evaluated after injection of Atlantic salmon for infection dynamics, genetic stability, transmission, and protection against a subsequent SAV3 challenge. Results: Attenuated clones demonstrated reduced virulence, as indicated by lower viral RNA loads, diminished transmission to cohabitant fish, and minimal clinical symptoms compared to the virulent wild-type virus. The clones mutated in both capsid and E2 exhibited the most attenuation, observed as rapid clearing of the infection and showing little transmission, while the clone with glycoprotein E2 mutations displayed greater residual virulence but provided stronger protection, seen as reduced viral loads upon subsequent challenge with SAV3. Despite their attenuation, all viral clones caused significant reductions in weight gain. Conclusions: Despite promising attenuation and protection, this study highlights the trade-offs between virulence and immunogenicity in live vaccine design. Concerns over environmental risks, such as the shedding of genetically modified virus, necessitate further evaluation. Future efforts should optimize vaccine candidates to balance attenuation, immunogenicity, and minimal side effects. Full article

Background/Objectives: Somatic and genetic mutations in glutathione peroxidases (GPxs), including GPx7 and GPx8, have been linked to intellectual disability, microcephaly, and various tumors. GPx7 and GPx8 evolved the latest among the GPx enzymes and are present in the endoplasmic reticulum. Although lacking a glutathione binding domain, GPx7 and GPx8 possess peroxidase activity that helps the body respond to cellular stress. However, the protein mutations in these peroxidases remain relatively understudied. Methods: By elucidating the structural and stability consequences of missense mutations, this study aims to provide insights into the pathogenic mechanisms involved in different cancers, thereby aiding clinical diagnosis, treatment strategies, and the development of targeted therapies. We performed saturated computational mutagenesis to analyze 2926 and 3971 missense mutations of GPx7 and GPx8, respectively. Results: The results indicate that G153H and G153F in GPx7 are highly destabilizing, while E93M and W142F are stabilizing. In GPx8, N74W and G173W caused the most instability while S70I and S119P increased stability. Our analysis shows that highly destabilizing somatic and genetic mutations are more likely pathogenic compared to stabilizing mutations. Conclusions: This comprehensive analysis of missense mutations in GPx7 and GPx8 provides critical insights into their impact on protein structure and stability, contributing to a deeper understanding of the roles of somatic mutations in cancer development and progression. These findings can inform more precise clinical diagnostics and targeted treatment approaches for cancers. Full article

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein (RBP) that plays a multifunctional role in RNA metabolism. TIA1 has three RNA-Recognition Motifs (RRMs) and a prion-like carboxyl C-terminal domain (LCD) with intrinsically disordered regions (IDR) implicated in the dynamics (i.e., formation, assembly, and disassembly) of transient RNA-protein aggregates known as stress granules (SGs). A protein related to TIA1 is its paralog TIA1-related/like protein (TIAR/TIAL1), whose amino acid sequence, structural organisation, and molecular and cellular functions are highly conserved with TIA1. Both proteins are the main components of SGs, which are non-membranous RNA-protein condensates formed under stress to promote cell survival. Welander distal myopathy (WDM) is a late-onset muscular dystrophy that has been linked to a single-nucleotide substitution (c.1362G>A; p.E384K) in the gene encoding the TIA1 protein, which impacts TIA1-dependent SGs dynamics. Herein, we have analysed cellular and molecular aspects by targeting mutagenesis to position 384 to understand its molecular grammar in an amino acid/proteinogenic-dependent or -independent manner under oxidative stress. The observations suggest differential, even opposing, behaviours between TIA1 and TIAR in the presence of specific amino acids with negative and positive charges, and also uncharged acids, at equivalent positions of TIA1 and TIAR, respectively. Collectively, these findings illustrate a characteristic molecular grammar of TIAR- and TIA1-dependent SGs under oxidative conditions, suggesting a gain of versatility between two structurally and functionally highly conserved/related proteins. Full article

Adeno-associated viral (AAV) vector-mediated gene therapy has emerged as a promising alternative to liver transplantation for monogenic metabolic hepatic diseases. AAVs are non-integrative vectors that are maintained primarily as episomes in quiescent cells like adult hepatocytes. This quality, while advantageous from a safety perspective due to a decreased risk of insertional mutagenesis, becomes a disadvantage when treating dividing cells, as it inevitably leads to the loss of the therapeutic genome. This is a challenge for the treatment of hereditary liver diseases that manifest in childhood. One potential approach to avoid vector genome loss involves putting scaffold/matrix attachment regions (S/MARs) into the recombinant AAV (rAAV) genome to facilitate its replication together with the cellular genome. We found that the administration of AAVs carrying the human β-interferon S/MAR sequence to neonatal and infant mice resulted in the maintenance of higher levels of viral genomes. However, we also observed that its inclusion at the 3′ end of the mRNA negatively impacted its stability, leading to reduced mRNA and protein levels. This effect can be partially attenuated by incorporating nonsense-mediated decay (NMD)-inhibitory sequences into the S/MAR containing rAAV genome, whose introduction may aid in the development of more efficient and longer-lasting gene therapy rAAV vectors. Full article

Background/Objectives: The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1) is a type I integral membrane glycoprotein discovered in monkeys and humans as a HAV receptor. HAVCR1 contains an N-terminal immunoglobulin-like variable domain (IgV) followed by a mucin-like domain (Muc), a transmembrane domain, and a cytoplasmic tail with a canonical tyrosine kinase phosphorylation site. The IgV binds phosphatidylserine on apoptotic cells, extracellular vesicles, and enveloped viruses. Insertions/deletions at position 156 (156ins/del) of the Muc were associated in humans with susceptibility to atopic, autoimmune, and infectious diseases. However, the molecular basis for the differential function of the HAVCR1 variants is not understood. Methods: We used mutagenesis, apoptotic cell binding, and signal transduction analyses to study the role of the 156ins/del in the function of HAVCR1. Results: We found that the HAVCR1 variant without insertions at position 156 (156delPMTTTV, or short-HAVCR1) bound more apoptotic cells than that containing a six amino acid insertion (156insPMTTTV, or long-HAVCR1). Furthermore, short-HAVCR1 induced stronger cell signaling and phagocytosis than long-HAVCR1. Conclusions: Our data indicated that the 156ins/del determine how the IgV is presented at the cell surface and modulate HAVCR1 binding, signaling, and phagocytosis, suggesting that variant-specific targeting could be used as therapeutic interventions to treat immune and infectious diseases. Full article

Cystathionine γ-lyase (CSE) is the second enzyme in the trans-sulfuration pathway that converts cystathionine to cysteine. It is also one of three major enzymes responsible for the biosynthesis of hydrogen sulfide (H₂S). CSE is believed to be the major source of endogenous H₂S in the cardiovascular system, and the CSE/H₂S system plays a crucial role in a variety of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is less well understood, especially at the post-translational level. Here, we demonstrated that the persulfidation of CSE inhibits its activity by ~2-fold in vitro. The loss of this post-translational modification in the presence of dithiothreitol (DTT) results in a reversal of basal activity. Cys137 was identified as the site for persulfidation by combining mass spectrometry, mutagenesis, activity analysis and streptavidin–biotin pull-down assays. To test the physiological relevance of the persulfidation regulation of CSE, human aortic vascular smooth muscle cells (HA-VSMCs) were incubated with vascular endothelial growth factor (VEGF), which is known to enhance endogenous H₂S levels. Under these conditions, consistent with the change tendency of the cellular H₂S level, the CSE persulfidation levels increased transiently and then gradually decreased to the basal level. Collectively, our study revealed a negative feedback regulation mechanism of the CSE/H₂S system via the persulfidation of CSE and demonstrated the potential for maintaining cellular H₂S homeostasis under oxidative stress conditions, particularly in tissues where CSE is a major source of H₂S. Full article

Trehalose is crucial for the stress resistance of Saccharomyces cerevisiae, primarily through its stabilization of proteins and membranes. The Agt1 transporter, a member of the Major Facilitator Superfamily, mediates trehalose uptake, a key process for maintaining cellular integrity under stress. Despite its importance, the molecular mechanisms of Agt1-mediated trehalose transport remain underexplored. In this study, we expressed and purified the trehalase enzyme TreA from E. coli to develop reliable trehalose assays. We screened 257 wild S. cerevisiae isolates, identifying strains with enhanced trehalose transport capacities. Comparative analyses, including structural modeling and molecular docking, revealed that specific Agt1 variants exhibited significantly higher transport efficiency, influenced by key residues in the transporter. Molecular dynamics simulations and steered molecular dynamics provided further insights, particularly into the role of the Agt1 channel head region in substrate recognition and binding. Site-directed mutagenesis validated these findings, showing that mutations at critical residues, such as 156Q, 164L, 256Q, 395E, 396R, and 507Y significantly reduced transport activity, while 137Q, 230T, and 514 N increased efficiency under certain conditions. Full article