Oxalyl-CoA synthetase from is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however...Oxalyl-CoA synthetase from is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.
Lipids function as the major building blocks of cellular membranes, as signaling molecules and as energy stores for metabolism. These important functions require a precise regulation of lipid biosynthesis, transport, tur...Lipids function as the major building blocks of cellular membranes, as signaling molecules and as energy stores for metabolism. These important functions require a precise regulation of lipid biosynthesis, transport, turnover and storage. Lipids are exchanged among organelles through a sophisticated network of vesicular and non-vesicular transport routes. Lysosomes, as the main catabolic organelle, are at the center of this network and have recently evolved as one of the master-regulators of cellular lipid metabolism. Lipids from both endogenous and exogenous sources can be processed, sensed and sorted in and out of the lysosome. In this review, we focus on the role of the lysosome in lipid catabolism, transport and signaling. We highlight recent discoveries on the transport of lipids out of the lysosomal lumen and their exchange with other organelles via membrane contact sites. We also discuss the direct role of lysosomal lipids in the TORC1 signaling pathway, a regulator of cellular metabolism. Finally, we address lysosomal biogenesis, its role in the sorting of lipid metabolic enzymes and the dysregulation of these processes in disease.
Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. Herein, we focus on Neprilysin 4 from and evaluate the existing knowledge on the physiologic...Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. Herein, we focus on Neprilysin 4 from and evaluate the existing knowledge on the physiological relevance of the peptidase. Particular attention is paid to the role of the neprilysin in regulating feeding behavior and the expression of insulin-like peptides in the central nervous system. In addition, we assess the function of the peptidase in controlling the activity of the sarcoplasmic and endoplasmic reticulum Ca ATPase in myocytes, as well as the underlying molecular mechanism in detail.
The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and t...The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue's function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein-Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.
Increase evidence from epidemiological studies have shown an inverse association between Parkinson's disease (PD) and lung cancer. PD and lung cancer are both geriatric diseases, where these two diseases are sharing some...Increase evidence from epidemiological studies have shown an inverse association between Parkinson's disease (PD) and lung cancer. PD and lung cancer are both geriatric diseases, where these two diseases are sharing some common genetic determinants. Several PD-associated genes including alpha synuclein (SNCA), PTEN-induced kinase 1 (PINK1), parkin, parkinsonism associated deglycase (DJ-1), leucine-rich repeat kinase 2 (LRRK2), F-box protein 7 (FBXO7) and ubiquitin C-terminal hydrolase L1 (UCHL1) were reported to have altered expressions in lung cancer patients. This indicates that certain PD-associated genes might be important in conferring anticancer effects. This review aims to depict the physiological functions of these genes, and discuss the putative roles of these PD-associated genes in lung cancer. The understanding of the roles of these genes in the lung cancer progression might be important in the identification of new treatment targets for lung cancer. Gene therapy that aims to alter the expressions of these genes could be developed for future anticancer therapy. As a result, studying the roles of these genes in lung cancer may also help to understand their involvements as well as their roles in the pathogenesis of PD.
Dipeptidyl peptidases constitute a class of non-classical serine proteases that regulate an array of biological functions, making them pharmacologically attractive enzymes. With this work, we identified and characterized...Dipeptidyl peptidases constitute a class of non-classical serine proteases that regulate an array of biological functions, making them pharmacologically attractive enzymes. With this work, we identified and characterized a dipeptidyl peptidase from (MtDPP) displaying a strong preference for proline residues at the P substrate position and an unexpectedly high thermal stability. MtDPP was also characterized with alanine replacements of residues of its active site that yielded, for the most part, loss of catalysis. We show that MtDPP catalytic activity is inhibited by well-known human DPP4 inhibitors. Using MALDI-TOF mass spectrometry we also describe that , MtDPP mediates the truncation of the C-X-C motif chemokine ligand 10, indicating a plausible role in immune modulation for this mycobacterial enzyme.
-Methyl--aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The developme...-Methyl--aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons.
Due to its unique features amongst ionotropic glutamate receptors, the NMDA receptor is of special interest in the physiological context but even more as a drug target. In the pathophysiology of metabolic disorders, part...Due to its unique features amongst ionotropic glutamate receptors, the NMDA receptor is of special interest in the physiological context but even more as a drug target. In the pathophysiology of metabolic disorders, particularly type 2 diabetes mellitus, there is evidence that NMDA receptor activation contributes to disease progression by impairing beta cell function. Consequently, channel inhibitors are suggested for treatment, but up to now there are many unanswered questions about the signaling pathways NMDA receptors are interfering with in the islets of Langerhans. In this review we give an overview about channel structure and function with special regard to the pancreatic beta cells and the regulation of insulin secretion. We sum up which signaling pathways from brain research have already been transferred to the beta cell, and what still needs to be proven. The main focus is on the relationship between an over-stimulated NMDA receptor and the production of reactive oxygen species, the amount of which is crucial for beta cell function. Finally, pilot studies using NMDA receptor blockers to protect the islet from dysfunction are reviewed and future perspectives for the use of such compounds in the context of impaired glucose homeostasis are discussed.
Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some c...Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some curvature-sensing molecules not only detect membrane deformation but can also alter the amplitude of forces that caused to shape changes in the first place. This dual ability of sensing and inducing membrane deformation leads to the formation of curvature-dependent self-organizing signaling circuits. How these cell-autonomous circuits are affected by auxiliary parameters from inside and outside of the cell has remained largely elusive. Here, we explore how such factors modulate self-organization at the micro-scale and its emerging properties at the macroscale.
Cancer is characterized by uncontrolled growth, invasion, and metastasis. In addition to solid cancer cells, cancer-associated fibroblasts (CAFs) play important roles in cancer pathophysiology. They arise from "healthy"...Cancer is characterized by uncontrolled growth, invasion, and metastasis. In addition to solid cancer cells, cancer-associated fibroblasts (CAFs) play important roles in cancer pathophysiology. They arise from "healthy" cells but get manipulated by solid cancer cells to supply them and develop a tumor microenvironment (TME) that protects the cancer cells from the immune defense. A wide variety of cell types can differentiate into CAFs, including fibroblasts, endothelial cells, and epithelial cells. Precise Ca regulation is essential for each cell including CAFs. The electrogenic Na/Ca exchanger (NCX) is one of the ubiquitously expressed regulatory Ca transport proteins that rapidly responds to changes of the intracellular ion concentrations. Its transport function is also influenced by the membrane potential and thereby indirectly by the activity of ion channels. NCX transports Ca out of the cell (forward mode) or allows its influx (reverse mode), always in exchange for 3 Na ions that are moved into the opposite direction. In this review, we discuss the functional roles NCX has in CAFs and how these depend on the properties of the TME. NCX activity modifies migration and leads to a reduced proliferation and apoptosis. The effect of the NCX in fibrosis is still largely unknown.
Ion channels play an important role for regulation of the exocrine and the endocrine pancreas. This review focuses on the Ca-regulated K channel K3.1, encoded by the gene, which is present in both parts of the pancreas....Ion channels play an important role for regulation of the exocrine and the endocrine pancreas. This review focuses on the Ca-regulated K channel K3.1, encoded by the gene, which is present in both parts of the pancreas. In the islets of Langerhans, K3.1 channels are involved in the regulation of membrane potential oscillations characterizing nutrient-stimulated islet activity. Channel upregulation is induced by gluco- or lipotoxic conditions and might contribute to micro-inflammation and impaired insulin release in type 2 diabetes mellitus as well as to diabetes-associated renal and vascular complications. In the exocrine pancreas K3.1 channels are expressed in acinar and ductal cells. They are thought to play a role for anion secretion during digestion but their physiological role has not been fully elucidated yet. Pancreatic carcinoma, especially pancreatic ductal adenocarcinoma (PDAC), is associated with drastic overexpression of K3.1. For pharmacological targeting of K3.1 channels, we are discussing the possible benefits K3.1 channel inhibitors might provide in the context of diabetes mellitus and pancreatic cancer, respectively. We are also giving a perspective for the use of a fluorescently labeled derivative of the K3.1 blocker senicapoc as a tool to monitor channel distribution in pancreatic tissue. In summary, modulating K3.1 channel activity is a useful strategy for exo-and endocrine pancreatic disease but further studies are needed to evaluate its clinical suitability.
For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different gene...For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.
The endolysosomal system of eukaryotic cells has a key role in the homeostasis of the plasma membrane, in signaling and nutrient uptake, and is abused by viruses and pathogens for entry. Endocytosis of plasma membrane pr...The endolysosomal system of eukaryotic cells has a key role in the homeostasis of the plasma membrane, in signaling and nutrient uptake, and is abused by viruses and pathogens for entry. Endocytosis of plasma membrane proteins results in vesicles, which fuse with the early endosome. If destined for lysosomal degradation, these proteins are packaged into intraluminal vesicles, converting an early endosome to a late endosome, which finally fuses with the lysosome. Each of these organelles has a unique membrane surface composition, which can form segmented membrane microcompartments by membrane contact sites or fission proteins. Furthermore, these organelles are in continuous exchange due to fission and fusion events. The underlying machinery, which maintains organelle identity along the pathway, is regulated by signaling processes. Here, we will focus on the Rab5 and Rab7 GTPases of early and late endosomes. As molecular switches, Rabs depend on activating guanine nucleotide exchange factors (GEFs). Over the last years, we characterized the Rab7 GEF, the Mon1-Ccz1 (MC1) complex, and key Rab7 effectors, the HOPS complex and retromer. Structural and functional analyses of these complexes lead to a molecular understanding of their function in the context of organelle biogenesis.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread all over the world. In this respect, traditional medicinal chemistry, repurposing, and computational approaches have been exploited to d...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread all over the world. In this respect, traditional medicinal chemistry, repurposing, and computational approaches have been exploited to develop novel medicines for treating this condition. The effectiveness of chemicals and testing methods in the identification of new promising therapies, and the extent of preparedness for future pandemics, have been further highly advantaged by recent breakthroughs in introducing noble small compounds for clinical testing purposes. Currently, numerous studies are developing small-molecule (SM) therapeutic products for inhibiting SARS-CoV-2 infection and replication, as well as managing the disease-related outcomes. Transmembrane serine protease (TMPRSS2)-inhibiting medicinal products can thus prevent the entry of the SARS-CoV-2 into the cells, and constrain its spreading along with the morbidity and mortality due to the coronavirus disease 2019 (COVID-19), particularly when co-administered with inhibitors such as chloroquine (CQ) and dihydroorotate dehydrogenase (DHODH). The present review demonstrates that the clinical-stage therapeutic agents, targeting additional viral proteins, might improve the effectiveness of COVID-19 treatment if applied as an adjuvant therapy side-by-side with RNA-dependent RNA polymerase (RdRp) inhibitors.
Amino acids play essential role for humans. We studied the dielectric properties of the basic aliphatic amino acids and polar positive amino acids in solutions of different concentrations. The high-Q single microwave whi...Amino acids play essential role for humans. We studied the dielectric properties of the basic aliphatic amino acids and polar positive amino acids in solutions of different concentrations. The high-Q single microwave whispering gallery-mode (WGM) quartz dielectric resonator based technique, enhanced to a number of measurement frequencies, was applied. The technique allows liquid investigation of micro- to nano- liter volumes filled in microfluidic channel on six discrete frequencies in the 30-40 GHz range. The dependencies of the complex permittivity on the molar mass show almost linear behavior for aliphatic amino acids at different concentrations. The study results are in good agreement with the calculated data obtained by Cole-Cole equation.
It is known that the thalamus plays an important role in pathological brain conditions involved in demyelinating, inflammatory and neurodegenerative diseases such as Multiple Sclerosis (MS). Beside immune cells and cytok...It is known that the thalamus plays an important role in pathological brain conditions involved in demyelinating, inflammatory and neurodegenerative diseases such as Multiple Sclerosis (MS). Beside immune cells and cytokines, ion channels were found to be key players in neuroinflammation. MS is a prototypical example of an autoimmune disease of the central nervous system that is classified as a channelopathy where abnormal ion channel function leads to symptoms and clinical signs. Here we review the influence of the cytokine-ion channel interaction in the thalamocortical system in demyelination and inflammation.
The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal target...The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) in the cytosol, the PEX5/cargo complex docks at the peroxisomal membrane by binding to PEX14. The PEX14 N-terminal domain (NTD) recognizes (di)aromatic peptides, mostly corresponding to Wxxx(F/Y)-motifs, with nano-to micromolar affinity. Human PEX5 possesses eight of these conserved motifs distributed within its 320-residue disordered N-terminal region. Here, we combine biophysical (ITC, NMR, CD), biochemical and computational methods to characterize the recognition of these (di)aromatic peptides motifs and identify key features that are recognized by PEX14. Notably, the eight motifs present in human PEX5 exhibit distinct affinities and energetic contributions for the interaction with the PEX14 NTD. Computational docking and analysis of the interactions of the (di)aromatic motifs identify the specific amino acids features that stabilize a helical conformation of the peptide ligands and mediate interactions with PEX14 NTD. We propose a refined consensus motif ΦΦ for high affinity binding to the PEX14 NTD and discuss conservation of the (di)aromatic peptide recognition by PEX14 in other species.
The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid...The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 (()-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs - of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters - showed higher lipophilicity (higher log values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters - were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters -. The isopropyl ester showed a promising log value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.