Neuronal function relies on precise compartmentalization into dendritic, somatic, axonal, and synaptic domains that require specialized cellular architectures. Axons, in particular, can extend over extraordinary distance...Neuronal function relies on precise compartmentalization into dendritic, somatic, axonal, and synaptic domains that require specialized cellular architectures. Axons, in particular, can extend over extraordinary distances while preserving stable membrane composition, mechanical integrity, and reliable excitability. A submembranous scaffold composed of spectrin, ankyrin, actin, and associated proteins provides a conserved platform for coupling membrane proteins to cytoskeletal support and organizing membrane domains. In axons, this scaffold assembles into a membrane-associated periodic skeleton (MPS) with near-regular spacing that supports mechanical load, patterns membrane components, and contributes to compartmental boundaries. Comparative genetics indicates that core principles of spectrin-ankyrin organization are ancient, whereas vertebrate evolution expanded spectrin and ankyrin families and enabled specialized excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. Pathogenic variants in spectrin and ankyrin genes disrupt neuronal development and excitability and cause a growing spectrum of neurodevelopmental and neurodegenerative disorders, underscoring scaffold integrity as a key determinant of circuit stability. Here, we examine how conserved spectrin-ankyrin scaffold principles were adapted to neuronal cell-type diversity and domain specialization, with emphasis on axons, synapses, and disease, and we discuss how the membrane-associated periodic skeleton may function not only as a stabilizing framework but also as a nanoscale organizer of neuronal membrane architecture.
Golgi-associated membrane scaffolds, or tethers, have broad roles in membrane-bound protein and lipid trafficking and in maintaining Golgi architecture. Accordingly, they exert strong influence over cellular development,...Golgi-associated membrane scaffolds, or tethers, have broad roles in membrane-bound protein and lipid trafficking and in maintaining Golgi architecture. Accordingly, they exert strong influence over cellular development, signalling, cargo modification and transport. An ever-expanding group of Golgins and multi-subunit tethering complexes assumes distinct functions in specific Golgi subcompartments in close partnership with Rab and ARL family GTPases. Their dysregulation or mutation impairs glycosylation, vesicle trafficking, and cytoskeletal dynamics, thereby contributing to a spectrum of human pathologies ranging from neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease) to cancers (e.g. lung, breast, colon) and metabolic defects (impaired insulin secretion and lipid droplet formation). Here, we review these diverse roles across molecular, cellular and organismal physiology.
Proteins of the Ras-family are guanine nucleotide binding proteins (GNBPs) involved in a variety of fundamental cellular processes, including cell proliferation, cell differentiation, cytoskeleton dynamics, vesicular pro...Proteins of the Ras-family are guanine nucleotide binding proteins (GNBPs) involved in a variety of fundamental cellular processes, including cell proliferation, cell differentiation, cytoskeleton dynamics, vesicular processes and intracellular transport. A dysregulation of Ras-signaling has been found to be causative for the development of diseases, such as diverse cancer types, RASopathies, neurodegenerative diseases and ciliopathies. Ras-proteins cycle between a GTP-bound on-state and a GDP-bound off-state. Ras-proteins show low intrinsic rates for nucleotide exchange and nucleotide hydrolysis. They need guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to accelerate both functions in order to act as true molecular switches in the physiological context. Ras-proteins and their regulators/effectors are targets of post-translational modifications (PTMs) such as phosphorylation, ac(et)ylation, lipidation and ubiquitination. These PTMs regulate their activity, subcellular localization and turnover. In a biological perspective, PTMs are essential components for cellular signaling cascades and for molecular pattern formation. Bacterial pathogens use PTMs of Ras-proteins to allow efficient infection processes. Besides, modifications of Ras-proteins were shown to be of therapeutic potential in oncogenic variants such as Ras G12C. In this review, we summarize current knowledge on Ras-signaling, while emphasizing PTMs as dynamic signaling hubs for its precise spatiotemporal control.
Cysteine cathepsins are papain-like proteases which play key roles in a large range of organisms, including apicomplexan parasites. These obligate endoparasites are responsible for many devastating diseases in human and/...Cysteine cathepsins are papain-like proteases which play key roles in a large range of organisms, including apicomplexan parasites. These obligate endoparasites are responsible for many devastating diseases in human and/or animal hosts, for which available treatments are limited or face the emergence of resistance. This review presents the cysteine cathepsins expressed by five major Apicomplexa (, , , and ), highlighting available data on their structures, specific or common features, and biological functions in the parasite biology and host-parasite interactions. Although they belong to the same phylum, apicomplexan parasites have very distinct life cycles and biology, which are well adapted to the hosts they infect and to the tissues within which they develop. Accordingly, apicomplexan cysteine cathepsins display a wide variety of functions, associated with shared (e.g., invasion of and egress from host cells) or unique (e.g., degradation of haemoglobin in ) biological pathways. Through their crucial functions and involvement in multiple parasite stages, these parasitic proteases represent assumed therapeutic targets. The description of apicomplexan cysteine cathepsins also appears uneven within the phylum, and further exploration of their biology and role is needed to drive novel preventive or curative intervention strategies.
Gentamicin (GM)-induced nephrotoxicity is closely linked to oxidative stress, with few therapeutic alternatives available. Chalcones are structurally versatile compounds with reported antioxidant properties, making them...Gentamicin (GM)-induced nephrotoxicity is closely linked to oxidative stress, with few therapeutic alternatives available. Chalcones are structurally versatile compounds with reported antioxidant properties, making them attractive candidates for nephroprotection. The 4'-aminochalcones substituted with -NO (31.25-15.62 µM) or -N(CH3) (7.81 µM) groups demonstrated significant protective effects, partially restoring (∼11 %) HK-2 cell viability after GM-induced injury. Both derivatives increased intracellular glutathione levels, reduced superoxide accumulation, and lowered cytoplasmic ROS concentration. Structural and electronic analyses revealed that the electron-donating group -N(CH3) enhanced antioxidant reactivity, whereas the electron-withdrawing group -NO decreased cytotoxicity. HK-2 cells were exposed to GM (IC = 5.0 ± 0.7 mmol/L) for 24 h, followed by treatment with 4'-aminochalcones (250-7.81 µM). Cell viability was assessed by MTT assay. ROS were quantified by flow cytometry using DCFH-DA, while redox balance was evaluated through glutathione content and superoxide accumulation. The electronic properties of chalcone derivatives were further investigated by density functional theory calculations. The 4'-aminochalcones mitigate GM-induced oxidative stress in renal cells, and their antioxidant and cytoprotective activities are strongly influenced by electronic substituents. Electron-donating groups enhance reactivity, whereas electron-withdrawing groups improve safety, underscoring the importance of structure-effect relationships in the design of chalcone-based nephroprotective agents.
CNKSR proteins are multidomain scaffold proteins that coordinate protein complex formation and regulate intracellular signaling via diverse protein-protein interactions. Here, we focus on CNKSR2 (also known as CNK2 or MA...CNKSR proteins are multidomain scaffold proteins that coordinate protein complex formation and regulate intracellular signaling via diverse protein-protein interactions. Here, we focus on CNKSR2 (also known as CNK2 or MAGUIN), which is encoded by one of three vertebrate CNKSR genes that have undergone diversification and specification over the course of evolution, since the emergence of the earliest CNKSR-like modular proteins, which are observed already in the genome sequences of Porifera (sponges). The vertebrate CNKSR2 protein is unique in that it is the only CNKSR scaffold molecule with substantial expression in the central nervous system, and also the only CNKSR scaffold molecule for which there is a clear causal link to disease; patients with mutations that affect the function of the CNKSR2 protein suffer from a neurodevelopmental seizure disorder. For this reason in particular, understanding the cellular signalling cascades that are regulated by the CNKSR2 protein are of essential importance. In this review, we introduce the CNKSR proteins with their evolutionary trajectory, followed by an overview of available data on the vertebrate CNKSR2, with the aim to provide a concise summary of what is known about the expression and function of CNKSR2 in neurons of the central nervous system.
Lipid-modified membrane-associated proteins can bind reversibly to cellular membranes, and their steady-state localization reflects a balance between membrane-bound and cytosolic pools. For many small GTPases of the Rho...Lipid-modified membrane-associated proteins can bind reversibly to cellular membranes, and their steady-state localization reflects a balance between membrane-bound and cytosolic pools. For many small GTPases of the Rho and Rab families, this balance is regulated by GDP dissociation inhibitors (GDIs), which control membrane association by shielding the prenyl group and coupling localization to the nucleotide state. In contrast, Ras proteins were long thought to lack a comparable regulatory system. The prenyl-binding protein PDE6D has emerged as a GDI-like factor for prenylated Ras proteins. Here, we discuss the role of PDE6D in KRAS trafficking and spatial organization, and examine its potential as a target for pharmacological inhibition of oncogenic KRAS signaling.
Guanylate-binding proteins (GBPs) are interferon-inducible large GTPases that play a central role in cell-autonomous immunity against intracellular bacterial pathogens. A defining feature of GBPs is their ability to tran...Guanylate-binding proteins (GBPs) are interferon-inducible large GTPases that play a central role in cell-autonomous immunity against intracellular bacterial pathogens. A defining feature of GBPs is their ability to translate GTP binding and hydrolysis into large-scale conformational rearrangements that drive self-assembly into higher-order structures, including dimers, polymers, and membrane-associated coatomers. This review integrates insights from biochemical, biophysical, structural, and cell biology studies to summarize current mechanistic models of GBP self-assembly, with a particular focus on human GBP1. We highlight how GTP hydrolysis-driven GBP1 polymers and coatomers act as self-regulating nanomachineries that recognize and remodel the pathogen-associated molecular pattern lipopolysaccharide, thereby fulfilling a dual function as immune sensor and effector in non-canonical inflammasome activation and bacterial membrane disruption. By directly linking nucleotide binding kinetics, enzymatic activity, and assembly dynamics to cellular and infection-related phenotypes, this review places decades of biochemical and biophysical work on GBP1 into a clear physiological context of antibacterial host defense.
RAS proteins control signals required for cell growth and survival and, when constitutively activated by mutation, can drive oncogenesis. RAS proteins are primarily regulated by their GTP or GDP binding state, which is c...RAS proteins control signals required for cell growth and survival and, when constitutively activated by mutation, can drive oncogenesis. RAS proteins are primarily regulated by their GTP or GDP binding state, which is controlled by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). RAS proteins are also substrates for dozens of posttranslational modifications (PTMs) that target them to membranes and serve as a secondary means of regulation. Because the newly developed direct RAS inhibitors do not produce durable responses in RAS-dependent cancer, there is renewed interest in targeting the PTMs of RAS. These modifications are the subject of this review.
Tricomplex inhibitors (TCIs) are a novel class of direct Ras inhibitors that target the GTP-bound Ras(on) state through recruitment of Cyclophilin A. Daraxonrasib (RMC-6236) is a pan-Ras TCI that was recently shown to re...Tricomplex inhibitors (TCIs) are a novel class of direct Ras inhibitors that target the GTP-bound Ras(on) state through recruitment of Cyclophilin A. Daraxonrasib (RMC-6236) is a pan-Ras TCI that was recently shown to restore GTPase activity of G12-mutant Ras proteins. Structural analysis of a pan-Ras TCI bound to K-Ras(GDP-AlF) reveals a transition-state arrangement of Tyr32 and Gln61 that closely resembles endogenous GTPase-GAP complexes. This includes a closed Switch-I conformation engaging the -GTPase machinery in a manner analogous to non-arginine-finger GAPs such as RanGAP. These observations position pan-Ras TCIs as pharmacologic GAP mimetics. The GTPase-promoting activity of daraxonrasib suggests synergy with Switch-II pocket K-Ras inhibitors, including the approved GDP-state selective K-Ras G12C inhibitor adagrasib (MRTX-849), whose engagement of K-Ras(GTP) is kinetically constrained by slow endogenous hydrolysis of the mutant GTPase. We demonstrate that daraxonrasib sensitizes K-Ras(GTP) to adagrasib labeling in both recombinant protein and cellular contexts. In K-Ras G12C and G12D mutant cell lines, combinations of daraxonrasib with adagrasib or HRS-4642 (MRTX-1133 analog) yield more rapid K-Ras engagement, rapid -ERK suppression, and significant Loewe synergy scores in viability assays. These findings establish GAP mimetics as rational and potent combination partners for Switch-II pocket inhibitors. The synergistic combination has potential to deepen and prolong pathway suppression while enabling dose reductions that may mitigate on-target toxicity and resistance.
Adherens junctions are cellular contact sites that organize epithelial tissues and play well-characterized roles in the coordination of cell collectives. Intercellular contacts are mediated by cadherin- or nectin-based a...Adherens junctions are cellular contact sites that organize epithelial tissues and play well-characterized roles in the coordination of cell collectives. Intercellular contacts are mediated by cadherin- or nectin-based adhesion and intracellularly linked to the actin cytoskeleton via the molecular scaffolds catenin and afadin. In this review, we discuss the mechanisms and roles of these molecular scaffolds for cellular morphogenesis and collective cell behaviour with a focus on neural tissue patterning. We discuss the molecular mechanisms in the conceptual framework of two often opposing, but complementary demands on adherens junctions in developing neural tissues: stability through 'supracellular' cytoskeletal linkage across cells versus local, dynamically adhesive cellular interactions in morphogenesis. Molecular scaffolds mediate localization, mechanosensitive adhesion and the regulation of cytoskeleton tension in both mechanistic contexts. These complementary mechanisms allow for collective behaviour that has predominantly been characterized for the patterning of tissues consisting of cell bodies, but was recently shown to also underlie the patterning of an epithelial-like tissue made entirely of neuronal growth cones. Molecular scaffolding of adherens junctions thereby contributes to patterning mechanisms that may apply to diverse tissue types.
Alfred Wittinghofer and his group of structural biologists and biochemists laid the groundwork for developing drugs that target RAS, from the first structure of a RAS protein in its active state, through to a deep unders...Alfred Wittinghofer and his group of structural biologists and biochemists laid the groundwork for developing drugs that target RAS, from the first structure of a RAS protein in its active state, through to a deep understanding of RAS function and regulation. Their fundamental discoveries include the mechanisms of hydrolysis and nucleotide exchange, effector engagement and subcellular localization. In this review, I share some of the highlights of years of a friendship and collaboration for the many years during which Fred's group led the field and helped understand and defeat the "beating heart of signal transduction".
The serine protease KLK7 contributes to several skin disorders and tumorigenesis, making it an attractive drug target. Owing to structural similarities in the S1 binding pocket between human KLK7 and certain trypsin-like...The serine protease KLK7 contributes to several skin disorders and tumorigenesis, making it an attractive drug target. Owing to structural similarities in the S1 binding pocket between human KLK7 and certain trypsin-like serine proteases harboring an Ala at position 190, compounds containing a chlorine-substituted benzylamide as P1 group were screened for KLK7 inhibition. Further optimization yielded the substrate analog inhibitor Bzls-d-hTyr-Pro-2-aminomethyl-5-chloro-benzylamide ( value 29.3 nM), which, however, had insufficient selectivity against the tested clotting proteases. Guided by a published KLK7 crystal structure, a second, more selective non-peptide inhibitor series was synthesized. The best derivatives ( values <100 nM) contain a chlorine-substituted aromatic P1 group and additionally address the non-prime region of KLK7. Murine Klk7 is poorly inhibited by these compounds; however, mutating threonine 190 in the S1 pocket to alanine restored the inhibitory potency. The new inhibitors effectively block KLK7-mediated functions, including chemokine cleavage and moesin gene upregulation, without cytotoxicity towards murine ovarian cancer cells. Moreover, tumors derived from ovarian cancer cells overexpressing either wild-type or mutated Klk7 reduced overall survival in mice compared to vector control cells. Together, these findings establish a robust inhibitor-enzyme system to evaluate human KLK7 inhibitors in a preclinical mouse model.
Recent studies have identified Aβ peptides in human gut epithelial cells, along with several amyloid-forming proteins and peptides in the gut lumen. These findings suggest that Aβ or other amyloid-like molecules originat...Recent studies have identified Aβ peptides in human gut epithelial cells, along with several amyloid-forming proteins and peptides in the gut lumen. These findings suggest that Aβ or other amyloid-like molecules originating from the gut may contribute to the involvement of the gastrointestinal system in the development of Alzheimer's disease (AD) pathology. Modulating the aggregation behaviour of Aβ and other amyloid forming peptides/proteins present in the gut may represent novel strategy to mitigate AD pathology. This study explores the use of Milk-derived Amyloid-like Protein Aggregates (MAPA) to inhibit Aβ(40) aggregation . MAPA's inhibitory effects were assessed using amyloid dye-binding assays (Thioflavin T, Congo Red, and ANS) and transmission electron microscopy. Toxicity assays showed that the MAPA significantly reduced Aβ(40)-induced neuronal death. Fluorescence quenching suggest MAPA physically interacts with Aβ(40) to prevent its aggregation. By blocking Aβ aggregation and reducing its neurotoxicity, MAPA presents a promising organic strategy to counteract AD progression influenced by gut factors. These findings open new avenues for AD prevention and the disease management, especially via dietary interventions targeting the gastro-intestinal system.
Albrecht Kossel (1853-1927) is associated with the discovery of the nucleobases adenine, guanine, thymine and cytosine, whose sequence in DNA forms genetic information. In 1910, he was awarded the Nobel Prize for his wor...Albrecht Kossel (1853-1927) is associated with the discovery of the nucleobases adenine, guanine, thymine and cytosine, whose sequence in DNA forms genetic information. In 1910, he was awarded the Nobel Prize for his work, which was foundational for biochemisry and the fields of nuclear biology. Kossel, who was editor of the predecessor of from 1895 to 1927, is hardly known today, either in Germany or among experts. The following is a tribute to his life and work on the occasion of the 100th anniversary of his death.
More than a century after Alois Alzheimer's neuropathological description, the mechanisms driving Alzheimer's disease (AD) remain only partially understood, and the failure of most clinical trials underscores the need to...More than a century after Alois Alzheimer's neuropathological description, the mechanisms driving Alzheimer's disease (AD) remain only partially understood, and the failure of most clinical trials underscores the need to identify and target alternative pathogenic pathways. Recent genetic, biochemical, and cellular studies support the view that AD is characterized by early alterations in the endolysosomal system and implicate multiple endocytic scaffold proteins as key drivers of AD progression. In this review, we summarize the current knowledge of five endocytic scaffold proteins, CALM, AP-2, BIN1, CD2AP, and ITSN1, which have been identified as AD risk factors by genome-wide association studies. We describe how, under physiological conditions, they couple membrane remodeling to intracellular signaling, whereas in AD they influence amyloid precursor protein trafficking, amyloid-β (Aβ) generation, tau pathology, and synaptic integrity. Finally, we propose a model in which cell type-specific and age-dependent dysfunction of endocytic scaffolds defines a pathogenic hotspot of proteostasis failure and offers new entry points for therapeutic intervention.
Intersectins with their numerous protein interaction domains serve as versatile scaffolds for a range of membrane-associated processes. While being originally characterized as endocytic proteins, their functions clearly...Intersectins with their numerous protein interaction domains serve as versatile scaffolds for a range of membrane-associated processes. While being originally characterized as endocytic proteins, their functions clearly extend beyond the scaffolding of endocytic machinery. By interacting with and stabilizing important cellular signaling components, ranging from neurotransmitter receptors to complexes involved in neuronal guidance, intersectins play a key role in shaping neuronal processes. Consequently, intersectin deficiency in model organisms causes the most severe impairments in the brain, illustrating their crucial function for neuronal development and neurotransmission. In line with this, mutations in the human gene encoding intersectin 1 (ITSN1) have been linked to neurodevelopmental and neuropsychiatric disorders. In addition, haploinsufficiency of ITSN1 has recently been associated with an increased risk of Parkinson´s disease. In this review, we will discuss our current knowledge regarding the molecular functions of intersectins in order to better understand the pathological consequences of intersectin deficiency.
In cardiomyocytes, the basic contractile unit are sarcomeres, which are organized in a regular manner facilitating their function. Here, we present a new computational approach to assess the functional properties of sarc...In cardiomyocytes, the basic contractile unit are sarcomeres, which are organized in a regular manner facilitating their function. Here, we present a new computational approach to assess the functional properties of sarcomeres at the nanoscale level in human cardiac cells, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We combined our analysis to different types of high-resolution imaging data, structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy-based imaging, as well as confocal microscopy data. We show that the radially averaged magnitude spectrum (RAMS) revealed sarcomere properties in a human cardiomyocyte model, iPSC-CMs, and compared our RAMS-based analysis to a real-space approach based on manually selected regions of interest. Moreover, we found the RAMS method suitable to quantify molecular differences of sarcomeres such as present in severe cardiac diseases, such as dilated cardiomyopathy (DCM). Defects in the sarcomere organization that occur in the presence of inherited DCM mutations in sarcomere proteins were efficiently recapitulated by our analysis. This new approach may facilitate streamlined analysis of molecular disease-specific phenotypic imaging data of cardiac cells, aiding our deeper understanding of the molecular basis of cardiac diseases.
The endolysosomal system connects Golgi and plasma membrane to the degradative pathway towards the lysosome and therefore presents a crossroads for endocytic recycling, secretory transport and degradation. This complexit...The endolysosomal system connects Golgi and plasma membrane to the degradative pathway towards the lysosome and therefore presents a crossroads for endocytic recycling, secretory transport and degradation. This complexity makes protein sorting and trafficking within the endolysosomal system challenging, and it requires tight regulation so that all proteins localize correctly. Proteins are sorted by distinct sorting adaptors, which recognize sorting signals and subsequently facilitate formation of transport carriers, which deliver content to other organelles. Alternatively, organelle maturation allows passive protein transport along different trafficking routes including endosomal and autophagosomal maturation. In this review, we will provide a bird's eye overview of the divers routes along which proteins are transported within the endolysosomal system and highlight open questions in the field.