Search Results (159)

Background: Diseases of veterinary importance, such as bovine Anaplasmosis, cause significant economic losses. Due to this, the study of various proteins of the causal agent Anaplasma marginale has focused on surface proteins. However, a vaccine for this disease is not yet available. To this end, in this work, moonlighting proteins (MLPs) are presented as an alternative approach for the design of immunogens against A. marginale. Methods: The proteins of the strain MEX-15-099-01 were analyzed, and its MLPs were identified. Subsequently, four virulence-associated MLP genes were selected and identified using PCR. The proteins were analyzed using a structural homology approach and the collection of B-cell epitopes was predicted for each MLP. Finally, a pair of AmEno peptides were synthesized and the antigenic potential was tested using an iELISA. Results: Our bioinformatics analysis revealed the potential of AmEno, AmGroEl, AmEF-Tu, and AmDnaK proteins as promising candidates for designing immunogens. The PCR allowed the gene sequence identification in the genome of the strain MEX-15-099-01. Notably, AmEno-derived synthetic peptides showed antigenicity in an ELISA. Conclusions: Our study has shed light on the potential use of MLPs for immunogen design, demonstrating the antigenic potential of AmEno. Full article

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K⁺ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K⁺ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding. Full article

This review describes the discovery, structure, activity, engineered constructs, and applications of KR-12, the smallest antibacterial peptide of human cathelicidin LL-37, the production of which can be induced under sunlight or by vitamin D. It is a moonlighting peptide that shows both antimicrobial and immune-regulatory effects. Compared to LL-37, KR-12 is extremely appealing due to its small size, lack of toxicity, and narrow-spectrum antimicrobial activity. Consequently, various KR-12 peptides have been engineered to tune peptide activity and stability via amino acid substitution, end capping, hybridization, conjugation, sidechain stapling, and backbone macrocyclization. We also mention recently discovered peptides KR-8 and RIK-10 that are shorter than KR-12. Nano-formulation provides an avenue to targeted delivery, controlled release, and increased bioavailability. In addition, KR-12 has been covalently immobilized on biomaterials/medical implants to prevent biofilm formation. These constructs with enhanced potency and stability are demonstrated to eradicate drug-resistant pathogens, disrupt preformed biofilms, neutralize endotoxins, and regulate host immune responses. Also highlighted are the safety and efficacy of these peptides in various topical and systemic animal models. Finaly, we summarize the achievements and discuss future developments of KR-12 peptides as cosmetic preservatives, novel antibiotics, anti-inflammatory peptides, and microbiota-restoring agents. Full article

Background: Accumulating evidence indicates that PSAT1 not only reprogrammed metabolic function but also exhibits “moonlighting” functions in promoting tumor malignancy. However, the underlying molecular mechanisms of PSAT1 promoting ER-negative breast cancer cell migration need further investigation. Methods: Briefly, the PSAT1 and ITGA2 expression in cells and tissues was detected using qRT-PCR, immunofluorescence staining and western blot assay. The effect of PSAT1 and ITGA2 was verified both in vitro and in vivo. RNA-seq analysis explored a series of differently expressed genes. The regulation between SP1 and ITGA2 was investigated by ChIP analysis. Results: We reported PSAT1 was highly expressed in ER-breast cancer tissues and tumor cells and positively correlated with metastasis. Moreover, RNA-seq analysis explored a series of differently expressed genes, including ITGA2, in PSAT1 overexpressed cells. Mechanistically, PSAT1 facilitated breast cancer metastasis via the p-AKT/SP1/ITGA2 axis. We further elucidated that PSAT1 promoted the entry of SP1 into the nucleus through the upregulation of p-AKT and confirmed ITGA2 is a target of SP1. In addition, enhanced cell migration was remarkably reversed by ITGA2 depletion or p-AKT inhibitor treatment. Conclusion: This study clarified the mechanism of PSAT1 in promoting ER-negative breast cancer metastasis, which may provide mechanistic clues for attenuating breast cancer metastasis. Full article

The water-soluble vitamin, vitamin B₁₂, also known as cobalamin, plays a crucial role in cellular metabolism, particularly in DNA synthesis, methylation, and mitochondrial functionality. Its deficiency can lead to hematological and neurological disorders; however, the manifestation of these clinical outcomes is relatively late. It leads to difficulties in the early diagnosis of vitamin B₁₂ deficiency. A prolonged lack of vitamin B₁₂ may have severe consequences including increased morbidity to neurological and cardiovascular diseases. Beyond inadequate dietary intake, vitamin B₁₂ deficiency might be caused by insufficient bioavailability, blood transport disruptions, or impaired cellular uptake and metabolism. Despite nearly 70 years of knowledge since the isolation and characterization of this vitamin, there are still gaps in understanding its metabolic pathways. Thus, this review aims to compile current knowledge about the crucial proteins necessary to efficiently accumulate and process vitamin B₁₂ in humans, presenting these systems as a multi-protein network. The epidemiological consequences, diagnosis, and treatment of vitamin B₁₂ deficiency are also highlighted. We also discuss clinical warnings of vitamin B₁₂ deficiency based on the ongoing test of specific moonlighting proteins engaged in vitamin B₁₂ metabolic pathways. Full article

Dysfunction of some mitochondrial aminoacyl-tRNA synthetases (encoded by the KARS1, HARS2, LARS2 and NARS2 genes) results in a great variety of phenotypes ranging from non-syndromic hearing impairment (NSHI) to very complex syndromes, with a predominance of neurological signs. The diversity of roles that are played by these moonlighting enzymes and the fact that most pathogenic variants are missense and affect different domains of these proteins in diverse compound heterozygous combinations make it difficult to establish genotype–phenotype correlations. We used a targeted gene-sequencing panel to investigate the presence of pathogenic variants in those four genes in cohorts of 175 Spanish and 18 Colombian familial cases with non-DFNB1 autosomal recessive NSHI. Disease-associated variants were found in five cases. Five mutations were novel as follows: c.766C>T in KARS1, c.475C>T, c.728A>C and c.1012G>A in HARS2, and c.795A>G in LARS2. We provide audiograms from patients at different ages to document the evolution of the hearing loss, which is mostly prelingual and progresses from moderate/severe to profound, the middle frequencies being more severely affected. No additional clinical sign was observed in any affected subject. Our results confirm the involvement of KARS1 in DFNB89 NSHI, for which until now there was limited evidence. Full article

Burkholderia cepacia complex infections remain life-threatening to cystic fibrosis patients, and due to the limited eradication efficiency of current treatments, novel antimicrobial therapies are urgently needed. Surface proteins are among the best targets to develop new therapeutic strategies since they are exposed to the host’s immune system. A surface-shaving approach was performed using Burkholderia cenocepacia J2315 to quantitatively compare the relative abundance of surface-exposed proteins (SEPs) expressed by the bacterium when grown under aerobic and microaerophilic conditions. After trypsin incubation of live bacteria and identification of resulting peptides by liquid chromatography coupled with mass spectrometry, a total of 461 proteins with ≥2 unique peptides were identified. Bioinformatics analyses revealed a total of 53 proteins predicted as localized at the outer membrane (OM) or extracellularly (E). Additionally, 37 proteins were predicted as moonlight proteins with OM or E secondary localization. B-cell linear epitope bioinformatics analysis of the proteins predicted to be OM and E-localized revealed 71 SEP moieties with predicted immunogenic epitopes. The protegenicity higher scores of proteins BCAM2761, BCAS0104, BCAL0151, and BCAL0849 point out these proteins as the best antigens for vaccine development. Additionally, 10 of the OM proteins also presented a high probability of playing important roles in adhesion to host cells, making them potential targets for passive immunotherapeutic approaches. The immunoreactivity of three of the OM proteins identified was experimentally demonstrated using serum samples from cystic fibrosis patients, validating our strategy for identifying immunoreactive moieties from surface-exposed proteins of potential interest for future immunotherapies development. Full article

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy. Full article

Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and can form protein–protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in the formation of the ribosome functional sites, such as the mRNA-binding site, tRNA-binding sites, the peptidyl transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. The extraribosomal activities of r-proteins have been studied over the decades. In the past decade, our understanding of r-protein functions has advanced significantly due to intensive studies on ribosomes and gene expression mechanisms not only in model bacteria like Escherichia coli or Bacillus subtilis but also in little-explored bacterial species from various phyla. The aim of this review is to update information on the multiple functions of r-proteins in bacteria. Full article

The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation–fibrinolytic system. Among the three main activation routes of complement, the lectin pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary (“moonlighting”) functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components. Full article

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed “moonlighting proteins”—proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this “moonlighting” role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins—vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10⁻⁸ M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall—agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)—were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively. Full article

WDR5 is a conserved nuclear protein that scaffolds the assembly of epigenetic regulatory complexes and moonlights in functions ranging from recruiting MYC oncoproteins to chromatin to facilitating the integrity of mitosis. It is also a high-value target for anti-cancer therapies, with small molecule WDR5 inhibitors and degraders undergoing extensive preclinical assessment. WDR5 inhibitors were originally conceived as epigenetic modulators, proposed to inhibit cancer cells by reversing oncogenic patterns of histone H3 lysine 4 methylation—a notion that persists to this day. This premise, however, does not withstand contemporary inspection and establishes expectations for the mechanisms and utility of WDR5 inhibitors that can likely never be met. Here, we highlight salient misconceptions regarding WDR5 inhibitors as epigenetic modulators and provide a unified model for their action as a ribosome-directed anti-cancer therapy that helps focus understanding of when and how the tumor-inhibiting properties of these agents can best be understood and exploited. Full article

Helminth parasites cause debilitating—sometimes fatal—diseases in humans and animals. Despite their impact on global health, mechanisms underlying host–parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization. Full article

Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins. Full article

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells’ self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells’ in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers. Full article