The phylum Euglenozoa, within the Eukaryote domain, includes diverse protists such as the medically significant kinetoplastids, characterized by their unique kinetoplast DNA. Both kinetoplastids and their sister class Diplonemea possess glycosomes - specialized microbodies that compartmentalize glycolysis and other metabolic pathways. Glycosomes likely evolved in a common ancestor of kinetoplastid and diplonemids, conferring metabolic flexibility and reducing cellular toxicity. These organelles are essential for parasite survival and thus, represent promising drug targets for treating kinetoplastid diseases. While the basic principles of peroxisome and glycosome biogenesis are conserved, distinct features in glycosome biogenesis machinery and a lower level of sequence conservation enables pathogen specific drug design for developing new therapies. This review summarizes our current knowledge on glycosome biogenesis, recent advances, and therapeutic potential for treating trypanosomatid infections.

The small protein family of VAMP-associated proteins (VAPs) have the unique position in cell biology as intracellular signposts for the Endoplasmic Reticulum (ER). VAP is recognised by a wide range of other proteins that use it to target the ER, either simply being recruited from the cytoplasm, or being recruited from separate organelles. The latter process makes VAP a component of many bridges between the ER and other compartments at membrane contact sites. The fundamental observations that identify VAP as the ER signpost have largely remained unchanged for over two decades. This review will describe how increased understanding of the special role of VAP in recent years has led to new discoveries: what constitutes the VAP family, how proteins bind to VAP, and which cellular functions connect to the ER using VAP. It will also describe the pitfalls that have led to difficulties determining how some proteins bind VAP and suggest some possibilities for future research.

Dendritic spines are the postsynaptic compartment of most excitatory synapses in the vertebrate brain. Their morphology is defined by a complex actin scaffold consisting of branched and unbranched actin filaments (F-actin), which constitute the major structural component of dendritic spines. During brain development, dendritic spines arise from dendritic filopodia, motile finger-like dendritic protrusions, whose morphology is also defined by an actin scaffold. The organization of the actin scaffold as well as its dynamic behavior in both dendritic filopodia and dendritic spines requires the coordinated activity of actin binding proteins (ABP) that promote either assembly or disassembly of F-actin. Studies of the past two decades identified a number of ABP and upstream regulatory pathways that control the morphology of dendritic spines as well as their morphological changes associated with synaptic plasticity, the cellular basis for learning and memory. Instead, much less is known about actin regulatory mechanisms that control the formation and elongation of dendritic filopodia or the structural changes associated with their transition into dendritic spines. This review article highlights recent advances in the field by summarizing and discussing studies of the past few years that provided exciting novel insights into the molecular machinery that governs dendritic filopodia initiation and their maturation into dendritic spines.

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.

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.

The 76th Mosbacher Kolloquium focused on recent advances in machine learning applications for structural biology and protein design. It covered topics spanning artificial intelligence-driven protein structure prediction, integrative modeling, generative protein design, and general applications in life sciences. With strong participation, high-caliber talks, and a clear focus on the integration of AI in biomolecular research, the meeting underscored the transformative role of machine learning in molecular biosciences and provided a vibrant platform for knowledge exchange across disciplines and generations.

Polyamines are ubiquitous and essential for cellular physiology, yet their metabolic pathways and functions remain only partially understood. Polyamine oxidases (PAO) are key to elucidating their physiological roles. In the methylotrophic yeast , we identified three putative PAO-encoding genes. Biochemical characterization showed that two of them function as PAOs, whereas the third has unknown substrate specificity. In contrast to previously studied yeasts, including , which contain only a single PAO, harbors multiple and functionally distinct PAOs. These findings highlight an unexpected diversification of polyamine catabolism in yeast and suggest previously unrecognized roles of PAOs in cellular physiology.

The rapid spread of bacterial resistance to antibiotics necessitates the development of innovative strategies to enhance their efficacy. One promising approach is incorporating antimicrobial peptides (AMPs) to synergize antibiotics. Herein, we introduce pH-responsive nanoplexes of plant AMP and sodium alginate (Na-Alg) for the co-delivery of AMP and Vancomycin (VCM) against resistant bacteria. The optimal nanoplexes (VCM-Na-Alg/AMP) were characterized, revealing a particle size, polydispersity index, zeta potential, encapsulation efficiency, and loading capacity of 159.5 ± 1.150 nm, 0.149 ± 0.018, -23.1 ± 0.1 mV, 82.34 ± 0.07 %, and 24.03 ± 0.10 % w/w, respectively. The nanoplexes exhibited pH-dependent changes in size and accelerated VCM release at acidic pH. antibacterial studies demonstrated a 2-fold enhanced activity against and methicillin-resistant (MRSA) and a 5-fold greater MRSA biofilm eradication, compared to bare VCM. Furthermore, the antibacterial activity evaluated on a mice model of MRSA systemic infection demonstrated that the nanoplexes reduced MRSA burden by 5-fold in kidneys and 4-fold in liver and blood. The nanoplexes also exhibited reduced inflammation and improved tissue integrity in the treated subjects. These findings present VCM-Na-Alg/AMP as a novel strategy to augment the efficacy of antibiotics against resistant bacteria.

Mitochondria are essential for cellular metabolism, serving as the primary source of adenosine triphosphate (ATP). This energy is generated by the oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Impairments in this machinery are linked to serious human diseases, especially in tissues with high energy demands. Assembly of the OXPHOS system requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes. The mitochondrial DNA encodes for 13 protein components, which are synthesized by mitochondrial ribosomes and inserted into the inner membrane during translation. Despite progress, key aspects of how mitochondrial gene expression is regulated remain elusive, largely due to the organelle's limited genetic accessibility. However, emerging technologies now offer new tools to manipulate various stages of this process. In this review, we explore recent strategies that expand our ability to target mitochondria genetically.

Plant exposure to unfavourable environmental conditions causes stress and reduces productivity. A common consequence of stress responses, are increased levels of reactive oxygen species (ROS), which if not controlled, could eventually lead to oxidative stress, damaging lipids, proteins and DNA, and ultimately result in cell death. One of the multiple defense systems that plants employ to regulate intracellular ROS levels are glutathione transferases (GSTs). GSTs have multiple roles in mitigating oxidative stress, e.g., by detoxifying xenobiotics through conjugation with reduced glutathione (GSH) or by using GSH to reduce damaging lipid hydroperoxides. In plants, GSTs exist in particularly large families and frequently occur in tandem gene clusters. This promotes the idea of functional diversification among closely related GSTs. This review focuses on the roles of GSTs in mitigating oxidative stress in plants and mentions potential strategies for functional analysis of the importance of individual GSTs by dissecting their enzymatic activities.

Stress responses in biological systems arise from complex, dynamic interactions among genes, proteins, and metabolites. A thorough understanding of these responses requires examining not only changes in individual molecular components but also their organization into interconnected pathways and networks that collectively maintain cellular homeostasis. This review provides an overview of computational strategies designed to capture these multifaceted processes. First, we discuss the importance of data analysis in uncovering how stress adaptation unfolds, highlighting both classical approaches (e.g., ANOVA, -tests) and more advanced methods (e.g., clustering, smoothing splines) that handle strong temporal dependencies. We then explore how enrichment analyses can contextualize these dynamic changes by linking regulated molecules to broader biological functions and processes. The latter half of the review focuses on network-based modeling techniques, emphasizing the construction and refinement of networks to identify stress-specific regulatory networks. Pairwise approaches are discussed alongside advanced methods that include multi-omics data, literature knowledge, and machine learning. Finally, we address comparative network analyses, which facilitate cross-condition studies, revealing both conserved and distinct features that shape resilience. With continued advances in high-throughput experimentation and computational modeling, these methods will deepen our insights into how cells detect and counteract stress.

Mitochondrial function relies heavily on the proper targeting and insertion of nuclear-encoded proteins into the outer mitochondrial membrane (OMM), a process mediated by specialised biogenesis factors known as insertases. These insertases are essential for the membrane integration of α-helical OMM proteins, which contain one or multiple hydrophobic transmembrane segments. While the general mechanisms of mitochondrial protein import are well established, recent research has shed light on the diversity and evolutionary conservation of OMM insertases across eukaryotic lineages. In , the mitochondrial import (MIM) complex, composed of Mim1 and Mim2, facilitates the integration of various α-helical OMM proteins, often in cooperation with import receptors such as Tom20 and Tom70. In , the functional MIM counterpart pATOM36 performs a similar role despite lacking sequence and structural homology, reflecting a case of convergent evolution. In mammals, MTCH2 has emerged as the principal OMM insertase, with MTCH1 playing a secondary, partially redundant role. This review provides a comparative analysis of these insertases, emphasising their conserved functionality, species-specific adaptations, and mechanistic nuances.

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.

The mitochondrial solute carrier family, also called SLC25 family, comprises a group of structurally and evolutionary related transporters that are embedded in the mitochondrial inner membrane. About 35 and 53 mitochondrial carrier proteins are known in yeast and human cells, respectively, which transport nucleotides, metabolites, amino acids, fatty acids, inorganic ions and cofactors across the inner membrane. They are proposed to function by a common rocker-switch mechanism, alternating between conformations that expose substrate-binding pockets to the intermembrane space (cytoplasmic state) and to the matrix (matrix state). The substrate specificities of both states differ so that carriers can operate as antiporters, symporters or uniporters. Carrier proteins share a characteristic structure comprising six transmembrane domains and expose both termini to the intermembrane space. Most carriers lack N-terminal presequences but use carrier-specific internal targeting signals that direct them into mitochondria via a specific import route, known as the 'carrier pathway'. Owing to their hydrophobicity and aggregation-prone nature, the mistargeting of carriers can lead to severe proteotoxic stress and diseases. In this review article, we provide an overview about the structure, biogenesis and physiology of carrier proteins, focusing on baker's yeast where their biology is particularly well characterized.

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.

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.

This study introduces a novel, rapid assay to measure CK2α activity in cell lysates. By fusing CK2α with the fluorescent protein mScarlet it was possible to quantify CK2α concentration directly in lysates. We used the dose-dependent increase of CK2α activity after addition of CK2β to determine the dissociation constants ( ) of the CK2α/CK2β-interaction. As a first trial, activity and affinity of the variant CK2α to CK2β was investigated using the developed assays. This mutation in the gene, encoding CK2α is related to the Okur-Chung Neurodevelopmental Syndrome (OCNDS). Apparent values of 13 nM for the CK2α/CK2β interaction and 7.4 nM for the CK2α/CK2β interaction were determined using nonlinear regression. Uncertainties with regards to the concentration of both binding partners were propagated through the entire process of nonlinear regression by Monte Carlo simulations. This way, accuracy confidence intervals of the -values were derived. This resulted in 96.4 % confidence that the accurate -values of the CK2α-CK2β and CK2α-CK2β interactions were different. The results suggest potential disruptions in oligomeric assembly caused by the R191Q mutation.