The application of synthetic riboswitches or aptamer-based biosensors for the monitoring of engineered metabolic pathways greatly depends on a high degree of target molecule specificity. Since metabolic pathways include...The application of synthetic riboswitches or aptamer-based biosensors for the monitoring of engineered metabolic pathways greatly depends on a high degree of target molecule specificity. Since metabolic pathways include close derivatives that often differ only in single moieties, the binding specificity of aptamers utilized for these systems has to be high. In the present study, we selected an RNA aptamer that is highly specific in its binding to homoeriodictyol while discriminating its close derivatives eriodictyol and naringenin. This high degree in specificity was achieved through three consecutive SELEX approaches while the selection parameters were adjusted and refined from one to the next. The adjustments along the process, with the selection outcome and next-generation sequencing analysis of the selection rounds, led to valuable insights into the stringency necessary to facilitate target specificity in aptamers obtained from SELEX. From the third selection, we obtained a highly binding specific aptamer and examined its structure and binding properties. Overall, our results connect the importance of selection stringency with SELEX outcome and aptamer specificity while providing a highly selective, homoeriodictyol-binding RNA aptamer.
The mitochondrial intermembrane space (IMS) houses proteins essential for redox regulation, protein import, signaling, and energy metabolism. Protein import into the IMS is mediated by dedicated pathways, including the d...The mitochondrial intermembrane space (IMS) houses proteins essential for redox regulation, protein import, signaling, and energy metabolism. Protein import into the IMS is mediated by dedicated pathways, including the disulfide relay pathway for oxidative folding. In addition, various IMS-traversing import pathways potentially expose unfolded proteins, representing threats to proteostasis. This trafficking of precursors coincides with unique biophysical challenges in the IMS, including a confined volume, elevated temperature, variable pH and high levels of reactive oxygen species. Ultrastructural properties and import supercomplex formation ameliorate these challenges. Nonetheless, IMS proteostasis requires constant maintenance by chaperones, folding catalysts, and proteases to counteract misfolding and aggregation. The IMS plays a key role in stress signaling, where proteostasis disruptions trigger responses including the integrated stress response (ISR) activated by mitochondrial stress (ISRmt) and responses to cytosolic accumulation of mitochondrial protein precursors. This review explores the biology and mechanisms governing IMS proteostasis, presents models, which have been employed to decipher IMS-specific stress responses, and discusses open questions.
has been used for a long time to treat autoimmune diseases. Its toxic side effects limit its clinical application. Mitophagy plays a protective role in various diseases. TANK-binding kinase 1 (TBK1) is a mitophagy-promot...has been used for a long time to treat autoimmune diseases. Its toxic side effects limit its clinical application. Mitophagy plays a protective role in various diseases. TANK-binding kinase 1 (TBK1) is a mitophagy-promoting molecule. This study aimed to investigate whether TBK1 could alleviate triptolide (TP)-induced nephrotoxicity by regulating mitophagy. To establish TP-induced nephrotoxic injury in animal model, 16 Sprague-Dawley rats were administered with TP by gavage, then renal tissues were collected for hematoxylin and eosin (HE) staining, western blotting and immunofluorescence analysis. To investigate whether up-regulation of TBK1 could alleviate TP-induced nephrotoxic injury and the specific mechanism, HK-2 cells were cultured , transfected with TBK1-overexpression recombinant lentivirus, then treated with TP. Western blotting, immunofluorescence, flow cytometry, multifunctional microplate detector were used to detect the relevant molecules. Here we found that TP caused kidney function damage, declined mitophagy levels, decreased the expression of TBK1 and mitophagy-related proteins in rats. TP stimulation decreased cell viability, mitochondrial membrane potential, mitophagy-protein, the formation of mito-autophagosomes and mito-autophagolysosomes in HK-2 cells. Upregulating TBK1 could reverse these damages. In summary, TP-induced cell injury had decreased mitophagy levels. Up-regulating TBK1 could increase mitophagy and further alleviate TP-induced cell injury.
Poirier-Bienvenu neurodevelopmental syndrome is a neurodevelopmental disorder associated with variants of the gene, characterized by intellectual disability, developmental delay, frequent seizures and more. While the m...Poirier-Bienvenu neurodevelopmental syndrome is a neurodevelopmental disorder associated with variants of the gene, characterized by intellectual disability, developmental delay, frequent seizures and more. While the majority of variants are nonsense variants leading to abortion of protein translation and no or truncated CK2β, many pathogenic missense variants also exist. We investigated the effect of four variants on CK2 holoenzyme formation and activity. We show that variants in the Zinc-finger region leads to reduced protein stability and altered subcellular localization. The instability is partly mediated by proteasomal and lysosomal degradation. We further show that homodimerization of these CK2β variants (p.Arg111Pro, p.Cys137Phe), localized within the Zinc-finger domain, is significantly reduced, while CK2α binding appears not affected. Other variants, p.Asp32Asn and p.Arg86Cys, did not affect stability or CK2β/α binding. For these mutants, the key to understanding the pathological mechanism may depend on external factors, such as altered protein-protein interaction. We conclude that Zinc-finger domain variants appear to destabilize the protein and affect holoenzyme formation, effectively reducing the pool of competent holoCK2. In the context of POBINDS, our findings suggest that Zinc-finger domain variants are likely to affect cells similarly to truncating and splicing variants with reduced translation of full-length CK2β.
Human papillomaviruses are causative agents in around 5 % of all cancers, and in a number of other human diseases. While prophylactic vaccines will alleviate the HPV disease burden on future generations, there are curren...Human papillomaviruses are causative agents in around 5 % of all cancers, and in a number of other human diseases. While prophylactic vaccines will alleviate the HPV disease burden on future generations, there are currently no therapeutic anti-viral strategies for combating HPV infections or lesions. HPV induce the proliferation of infected epithelial cells and modulate the host differentiation response, and both of these controls are required for a successful viral life cycle. Enhanced understanding of viral-host interactions during the viral life cycle will identify potential novel anti-viral strategies for therapeutic development. This minireview will summarize the critical role of the host enzyme CK2 in regulating the function of the viral proteins E1, E2 and E7; such control makes CK2 a critical enzyme for regulating HPV life cycles. Therapeutic strategies blocking CK2 function to combat HPV infections and treat HPV diseases will be described.
Proteome-wide scale in a dose- and time-depending setting is crucial to fully understand the pharmacological mechanism of anticancer drugs as well as identification of candidates for drug response biomarkers. Here, we in...Proteome-wide scale in a dose- and time-depending setting is crucial to fully understand the pharmacological mechanism of anticancer drugs as well as identification of candidates for drug response biomarkers. Here, we investigated the effect of the CIGB-300 anticancer peptide at IC and IC doses during 1 and 4 h of treatment on the squamous lung cancer cell (NCI-H226) proteome. An overwhelming dose-dependent inhibitory effect with minor up-regulated proteins was observed by increasing CIGB-300 dose level. Functional enrichment was also CIGB-300 dose-dependent with common or exclusively regulated proteins in each dose and time settings. A protein core involving small molecule biosynthesis, aldehyde metabolism and metabolism of nucleobases was regulated irrespectively to the dose or the treatment time. Importantly, a group of proteins linked to NSCLC tumor biology, poor clinical outcome and some Protein Kinase CK2 substrates, were significantly regulated by treating with both CIGB-300 doses. Likewise, we observed a consistent downregulation of different proteins that had been already reported to be inhibited by CIGB-300 in lung adenocarcinoma and acute myeloid leukemia. Overall, our proteomics-guided strategy based on time and drug dose served to uncover novel clues supporting the CIGB-300 cytotoxic effect and also to identify putative pharmacodynamic biomarkers in NSCLC.
The molecular chaperone Hsp90 is the central element of a chaperone machinery in the cytosol of eukaryotic cells that is characterized by a large number of structurally and functionally different co-chaperones that influ...The molecular chaperone Hsp90 is the central element of a chaperone machinery in the cytosol of eukaryotic cells that is characterized by a large number of structurally and functionally different co-chaperones that influence the core chaperone component in different ways and increase its influence on the proteome. From yeast to humans, the number of Hsp90 co-chaperones has increased from 14 to over 40, and new co-chaperones are still being discovered. While Hsp90 itself has only undergone limited changes in structure and mechanism from yeast to humans, its increased importance and contribution to different processes in humans is based on the evolution and expansion of the cohort of co-chaperones. In this review, we provide an overview of Hsp90 co-chaperones, focusing on their roles in regulating Hsp90 function and their evolution from yeast to humans.
Mitochondria are central hubs of cellular metabolism and their dysfunction has been implicated in a variety of human pathologies and the onset of aging. To ensure proper mitochondrial function under misfolding stress, a...Mitochondria are central hubs of cellular metabolism and their dysfunction has been implicated in a variety of human pathologies and the onset of aging. To ensure proper mitochondrial function under misfolding stress, a retrograde mitochondrial signaling pathway known as UPR is activated. The UPR ensures that mitochondrial stress is communicated to the nucleus, where gene expression for several mitochondrial proteases and chaperones is induced, forming a protective mechanism to restore mitochondrial proteostasis and function. Importantly, the UPR not only acts within cells, but also exhibits a conserved cell-nonautonomous activation across species, where mitochondrial stress in a defined tissue triggers a systemic response that affects distant organs. Here, we summarize the molecular basis of the UPR in the invertebrate model organism and in mammals. We also describe recent findings on cell-nonautonomous activation of the UPR in worms, flies and mice, and how UPR activation in specific tissues affects organismal metabolism and longevity.
Heat stress transcription factors (HSFs) play a pivotal role in regulating plant responses to heat and other environmental stresses, as well as developmental processes. HSFs possess conserved domains responsible for DNA...Heat stress transcription factors (HSFs) play a pivotal role in regulating plant responses to heat and other environmental stresses, as well as developmental processes. HSFs possess conserved domains responsible for DNA binding, oligomerization, and transcriptional regulation, which collectively enable precise and dynamic control of cellular responses to environmental stimuli. Functional diversification of HSFs has been demonstrated through genetic studies in model plants such as and economically important crops like tomato, rice, and wheat. However, the underlying molecular mechanisms that govern HSF function remain only partially understood, and for a handful of HSFs. Advancements in structural biology, biochemistry, molecular biology, and genomics shed light into how HSFs mediate stress responses at the molecular level. These insights offer exciting opportunities to leverage HSF biology for gene editing and crop improvement, enabling the customization of stress tolerance traits via regulation of HSF-dependent regulatory networks to enhance thermotolerance. This review synthesizes current knowledge on HSF structure and function, providing a perspective on their roles in plant adaptation to a changing climate.
Protein kinase CK2 (casein kinase 2) mainly exists as heterotetrameric holoenzyme with two catalytic subunits (CK2α or CK2α') bound to a homodimer of non-catalytic subunits (CK2β). With and , the human genome contains t...Protein kinase CK2 (casein kinase 2) mainly exists as heterotetrameric holoenzyme with two catalytic subunits (CK2α or CK2α') bound to a homodimer of non-catalytic subunits (CK2β). With and , the human genome contains two paralogs encoding catalytic CK2 subunits. Both gene products, called CK2α and CK2α', strongly interact with CK2β. An earlier report that CK2α' has a lower CK2β affinity than CK2α is confirmed via isothermal titration calorimetry in this study. Furthermore, we show with a fluorescence-anisotropy assay that a CK2β-competitive peptide binds less strongly to CK2α' than to CK2α. The reason for the reduced affinity of CK2α' to CK2β and CK2β competitors is puzzling: both isoenzymes have identical amino acid compositions at their CK2β interfaces, but the β4β5 loop, a component of this interface, is conformationally less adaptable in CK2α' than in CK2α due to intramolecular constraints. To release these constraints, we constructed a CK2α' mutant that was equalized to CK2α at the backside of the β4β5 loop. Concerning thermostability, affinity to CK2β or CK2β competitors and 3D-structure next to the β4β5 loop, this CK2α' mutant is more similar to CK2α than to its own wild-type, suggesting a critical role of the β4β5 loop adaptability for CK2β affinity.
Aneuploidy refers to chromosome number abnormality that is not an exact multiple of the haploid chromosome set. Aneuploidy has largely negative consequences in cells and organisms, manifested as so-called aneuploidy-asso...Aneuploidy refers to chromosome number abnormality that is not an exact multiple of the haploid chromosome set. Aneuploidy has largely negative consequences in cells and organisms, manifested as so-called aneuploidy-associated stresses. A major consequence of aneuploidy is proteotoxic stress due to abnormal protein expression from imbalanced chromosome numbers. Recent advances have improved our understanding of the nature of the proteostasis imbalance caused by aneuploidy and highlighted their relevance with respect to organellar homeostasis, dosage compensation, or mechanisms employed by cells to mitigate the detrimental stress. In this review, we highlight the recent findings and outline questions to be addressed in future research.
The correct synthesis of new proteins is essential for maintaining a functional proteome and cell viability. This process is tightly regulated, with ribosomes and associated protein biogenesis factors ensuring proper pro...The correct synthesis of new proteins is essential for maintaining a functional proteome and cell viability. This process is tightly regulated, with ribosomes and associated protein biogenesis factors ensuring proper protein production, modification, and targeting. In eukaryotes, the conserved nascent polypeptide-associated complex (NAC) plays a central role in coordinating early protein processing by regulating the ribosome access of multiple protein biogenesis factors. NAC recruits modifying enzymes to the ribosomal exit site to process the N-terminus of nascent proteins and directs secretory proteins into the SRP-mediated targeting pathway. In this review we will focus on these pathways, which are critical for proper protein production, and summarize recent advances in understanding the cotranslational functions and mechanisms of NAC in higher eukaryotes.
Mitochondrial functions and biogenesis depend on the import of more than 1,000 proteins which are synthesized as precursor proteins on cytosolic ribosomes. Mitochondrial protein translocases sort the precursor proteins i...Mitochondrial functions and biogenesis depend on the import of more than 1,000 proteins which are synthesized as precursor proteins on cytosolic ribosomes. Mitochondrial protein translocases sort the precursor proteins into the mitochondrial sub-compartments: outer and inner membrane, the intermembrane space and the matrix. The translocase of the outer mitochondrial membrane (TOM complex) constitutes the major import site for most of these precursor proteins. Defective protein translocases, premature folding of the precursor, or depletion of the membrane potential can cause clogging of the TOM channel by a precursor protein. This clogging impairs further protein import and leads to accumulation of precursor proteins in the cell that perturbates protein homeostasis, leading to proteotoxic stress in the cell. Therefore, unclogging of the translocon is critical for maintaining mitochondrial and cellular function. Ubiquitylation and AAA-ATPases play a central role in the extraction of the precursor proteins to deliver them to the proteasome for degradation. Here we summarize our understanding of the molecular mechanisms that remove such translocation-stalled precursor proteins from the translocation channel to regenerate the TOM complex for protein import.
Protein kinase CK2 is a promising therapeutic target, especially in oncology. Over the years, various inhibitors have been developed, with polyhalogenated scaffolds emerging as a particularly effective class. Halogens li...Protein kinase CK2 is a promising therapeutic target, especially in oncology. Over the years, various inhibitors have been developed, with polyhalogenated scaffolds emerging as a particularly effective class. Halogens like bromine and chlorine enhance inhibitor stability by forming additional interactions within the ATP pocket. Among halogenated scaffolds, benzotriazole and benzimidazole have led to potent molecules such as 4,5,6,7-tetrabromo-1-benzotriazole (IC = 300 nM) and 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole (IC = 140 nM). Modifications, including 4,5,6-tribromo-7-ethyl-1-benzotriazole (IC = 160 nM), further improved activity. Changing scaffolds while retaining halogens has enabled design of new inhibitors. Flavonols, dibenzofuranones, and the indeno[1,2-]indole scaffold are key examples. Halogenation of the reference molecule 5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-]indole-9,10-dione (, IC = 360 nM) significantly boosted potency. The study focused on introducing four halogens, yielding to the compound 1,2,3,4-tetrabromo-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-]indole-9,10-dione (), with an IC of 16 nM. Co-crystallography revealed how bromine atoms enhance binding, and demonstrated strong activity, particularly against leukemic cell lines like IPC-Bcl2.
The glycosylphosphatidylinositol (GPI)-anchored serine proteases, prostasin and testisin, have essential roles in diverse physiological functions including development, reproduction, homeostasis and barrier function of e...The glycosylphosphatidylinositol (GPI)-anchored serine proteases, prostasin and testisin, have essential roles in diverse physiological functions including development, reproduction, homeostasis and barrier function of epithelia, angiogenesis, coagulation, and fibrinolysis. Important functions in pathological conditions such as cancer, kidney disease and cardiovascular disease have also been reported. In this review, we summarize current knowledge of the cellular and roles of prostasin and testisin in physiology and pathophysiology and explore the underlying molecular mechanisms. We discuss how new insights of their role in cancer and cardiovascular disease may facilitate translation into clinical settings in the future.
The disruption of protein homeostasis leads to the increased un- and misfolding of proteins and the formation of toxic protein aggregates. Their accumulation triggers an unfolded protein response that is characterized by...The disruption of protein homeostasis leads to the increased un- and misfolding of proteins and the formation of toxic protein aggregates. Their accumulation triggers an unfolded protein response that is characterized by the transcriptional upregulation of molecular chaperones and proteases, and aims to restore proteome integrity, maintain cellular function, suppress the cause of perturbation, and prevent disease and death. In the green microalga , the study of this response to proteotoxic stress has provided insights into the function of chaperone and protease systems, which are, though simpler, closely related to those found in land plants. In addition, there has been considerable progress in understanding the triggers and regulation of compartment-specific unfolded protein responses. This review provides an overview on how the dysfunction of protein homeostasis is sensed in the different compartments of , and summarizes the current knowledge on the pathways that are triggered to restore equilibrium in the cell, while also highlighting similarities and differences to the unfolded protein responses of other model organisms.
Neurons are highly dependent on mitochondrial respiration for energy, rendering them vulnerable to oxidative stress. Reactive oxygen species (ROS), by-products of oxidative phosphorylation, can damage lipids, proteins, a...Neurons are highly dependent on mitochondrial respiration for energy, rendering them vulnerable to oxidative stress. Reactive oxygen species (ROS), by-products of oxidative phosphorylation, can damage lipids, proteins, and DNA, potentially triggering cell death pathways. This review explores the neuronal vulnerability to ROS, highlighting metabolic adaptations and antioxidant systems that mitigate oxidative damage. Balancing metabolic needs and oxidative stress defenses is critical for neurons, as disruptions are implicated in neurodegenerative diseases. Neurons uniquely modulate metabolic pathways, favoring glycolysis over oxidative phosphorylation in cell bodies, to minimize harmful ROS production. Key antioxidants, including superoxide dismutases and glutathione peroxidases, play crucial roles in neuronal protection, as evident from genetic studies linking deficiencies to neurodegeneration. Notably, neurons have the ability to adapt to oxidative conditions in compartment-specific manners and also utilize ROS as a signaling molecule to promote adaptive synaptic plasticity. Future research should aim to elucidate differential ROS signaling and antioxidant responses across neuronal compartments for improved therapeutic strategies.
Dipeptide production mediated by dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11 plays a crucial role in growth of , a periodontopathic asaccharolytic bacterium. Given the particular P1-position specificity of DPPs, i...Dipeptide production mediated by dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11 plays a crucial role in growth of , a periodontopathic asaccharolytic bacterium. Given the particular P1-position specificity of DPPs, it has been speculated that DPP5 or DPP7 might be responsible for degrading refractory P1 amino acids, , neutral (Thr, His, Gly, Ser, Gln) and hydrophilic (Asn) residues. The present results identified DPP7 as an entity that processes these residues, thus ensuring complete production of nutritional dipeptides in the bacterium. Activity enhancement by the P1' residue was observed in DPP7, as well as DPP4 and DPP5. Toward the refractory P1 residues, DPP7 uniquely hydrolyzed HX|LD-MCA (X = His, Gln, or Asn) and their hydrolysis was most significantly suppressed in gene-disrupted cells. Additionally, hydrophobic P2 residue significantly enhanced DPP7 activity toward these substrates. The findings propose a comprehensive 20 P1 × 20 P2 amino acid matrix showing the coordination of four DPPs to achieve complete dipeptide production along with subsidiary peptidases. The present finding of a broad substrate specificity that DPP7 accounts for releasing 48 % (192/400) of N-terminal dipeptides could implicate its potential role in linking periodontopathic disease to related systemic disorders.
In the early days of CK2 research, it was already published that the affinity of CK2 for its substrate casein was affected by insulin. Subsequent to the discovery of inhibitors of CK2 kinase activity, it was shown that C...In the early days of CK2 research, it was already published that the affinity of CK2 for its substrate casein was affected by insulin. Subsequent to the discovery of inhibitors of CK2 kinase activity, it was shown that CK2 has an influence on hormones that regulate glucose homeostasis and on enzymes that influence glucose metabolism in pancreatic islet cells as well as in hormone-sensitive target cells. This regulation includes the influence on transcription factors and thereby, gene expression, as well as direct modulation of the catalytic activity. The used CK2 inhibitors, especially the older ones, show a broad range of specificity, selectivity and off-target effects. Recently applied methods to down-regulate the expression of individual CK2 subunits using siRNA or CRISPR/Cas9 technology have contributed to the improvement of specificity. It was shown that inhibition of CK2 kinase activity or knock-down or knock-out of CK2α leads to an elevated synthesis and secretion of insulin in pancreatic β-cells and a down-regulation of the synthesis and secretion of glucagon from pancreatic α-cells. In the present review CK2-dependent molecular mechanisms will be addressed which contribute to the maintenance of glucose homeostasis.
Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into mitochondria. If the rate of protein synthesis exceeds the capacity of the mitochondrial import machinery, precursor prote...Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into mitochondria. If the rate of protein synthesis exceeds the capacity of the mitochondrial import machinery, precursor proteins can transiently accumulate in the cytosol. The cytosolic accumulation of mitochondrial precursors jeopardizes cellular protein homeostasis (proteostasis) and can be the cause of diseases. In order to prevent these toxic effects, most non-imported precursors are rapidly degraded by the ubiquitin-proteasome system. However, cells employ a second layer of defense which is the facilitated sequestration of mitochondrial precursor proteins in transient protein aggregates. The formation of such structures is triggered by nucleation factors such as small heat shock proteins. Disaggregases and chaperones can liberate precursors from cytosolic aggregates to pass them on to the mitochondrial import machinery or, under persistent stress conditions, to the proteasome for degradation. Owing to their role as transient buffering systems, these aggregates were referred to as MitoStores. This review articles provides a general overview about the MitoStore concept and the early stages in mitochondrial protein biogenesis in yeast and, in cases where aspects differ, in mammalian cells.