C-Glucosylation as a tool for the prevention of PAINS-induced membrane dipole potential alterations

AbstractThe concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols—phloretin, genistein and resveratrol—act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C–C ligation of sugars.

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Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

International Journal of Molecular Sciences ◽

10.3390/ijms22052732 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2732

Author(s):

Nadine Reichhart ◽

Vladimir M. Milenkovic ◽

Christian H. Wetzel ◽

Olaf Strauß

Keyword(s):

Transmembrane Protein ◽

Functional Characterization ◽

Missense Mutations ◽

Mutant Proteins ◽

Functional Consequences ◽

New Perspective ◽

The Stability ◽

Dynamics Simulations ◽

And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

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Ubiquitin and Ubiquitin-Like Proteins and Domains in Ribosome Production and Function: Chance or Necessity?

International Journal of Molecular Sciences ◽

10.3390/ijms22094359 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4359

Author(s):

Sara Martín-Villanueva ◽

Gabriel Gutiérrez ◽

Dieter Kressler ◽

Jesús de la Cruz

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Cellular Processes ◽

Substrate Protein ◽

Precursor Proteins ◽

Assembly Factors ◽

Ubiquitin Moiety ◽

And Function

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.

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Akt2 Regulates Cardiac Metabolism and Cardiomyocyte Survival

Journal of Biological Chemistry ◽

10.1074/jbc.m513087200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32841-32851 ◽

Cited By ~ 110

Author(s):

Brian DeBosch ◽

Nandakumar Sambandam ◽

Carla Weinheimer ◽

Michael Courtois ◽

Anthony J. Muslin

Keyword(s):

Pressure Overload ◽

Cardiac Metabolism ◽

Cellular Protein ◽

Growth Responses ◽

Infarct Zone ◽

Targeted Disruption ◽

Cellular Processes ◽

Ligand Stimulation ◽

And Function

The Akt family of serine-threonine kinases participates in diverse cellular processes, including the promotion of cell survival, glucose metabolism, and cellular protein synthesis. All three known Akt family members, Akt1, Akt2 and Akt3, are expressed in the myocardium, although Akt1 and Akt2 are most abundant. Previous studies demonstrated that Akt1 and Akt3 overexpression results in enhanced myocardial size and function. Yet, little is known about the role of Akt2 in modulating cardiac metabolism, survival, and growth. Here, we utilize murine models with targeted disruption of the akt2 or the akt1 genes to demonstrate that Akt2, but not Akt1, is required for insulin-stimulated 2-[3H]deoxyglucose uptake and metabolism. In contrast, akt2-/- mice displayed normal cardiac growth responses to provocative stimulation, including ligand stimulation of cultured cardiomyocytes, pressure overload by transverse aortic constriction, and myocardial infarction. However, akt2-/- mice were found to be sensitized to cardiomyocyte apoptosis in response to ischemic injury, and apoptosis was significantly increased in the peri-infarct zone of akt2-/- hearts 7 days after occlusion of the left coronary artery. These results implicate Akt2 in the regulation of cardiomyocyte metabolism and survival.

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The Role of the CX3CL1-CX3CR1 Axis in Renal Disease

AJP Renal Physiology ◽

10.1152/ajprenal.00059.2021 ◽

2021 ◽

Author(s):

Diana Hamdan ◽

Lisa A. Robinson

Keyword(s):

Therapeutic Potential ◽

Kidney Diseases ◽

Transmembrane Protein ◽

Soluble Form ◽

Protective Effects ◽

Chronic Kidney Diseases ◽

The Family ◽

Soluble Species ◽

And Function

Excessive infiltration of immune cells into the kidney is a key feature of acute and chronic kidney diseases. The family of chemokines are key drivers of this process. CX3CL1 (fractalkine) is one of two unique chemokines synthesized as a transmembrane protein which undergoes proteolytic cleavage to generate a soluble species. Through interacting with its cognate receptor, CX3CR1, CX3CL1 was originally shown to act as a conventional chemoattractant in the soluble form, and as an adhesion molecule in the transmembrane form. Since then, other functions of CX3CL1 beyond leukocyte recruitment have been described, including cell survival, immunosurveillance, and cell-mediated cytotoxicity. This review summarizes diverse roles of CX3CL1 in kidney disease and potential uses as a therapeutic target and novel biomarker. As the CX3CL1-CX3CR1 axis has been shown to contribute to both detrimental and protective effects in various kidney diseases, a thorough understanding of how the expression and function of CX3CL1 are regulated is needed to unlock its therapeutic potential.

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PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem

Development ◽

10.1242/dev.127.23.5157 ◽

2000 ◽

Vol 127 (23) ◽

pp. 5157-5165 ◽

Cited By ~ 13

Author(s):

T. Vernoux ◽

J. Kronenberger ◽

O. Grandjean ◽

P. Laufs ◽

J. Traas

Keyword(s):

Cell Fate ◽

Apical Meristem ◽

Transmembrane Protein ◽

Loss Of Function ◽

Hybrid Identity ◽

Hormone Transport ◽

Early Markers ◽

Ideal Tool ◽

And Function

The process of organ positioning has been addressed, using the pin-formed 1 (pin1) mutant as a tool. PIN1 is a transmembrane protein involved in auxin transport in Arabidopsis. Loss of function severely affects organ initiation, and pin1 mutants are characterised by an inflorescence meristem that does not initiate any flowers, resulting in the formation of a naked inflorescence stem. This phenotype, combined with the proposed role of PIN1 in hormone transport, makes the mutant an ideal tool to study organ formation and phyllotaxis, and here we present a detailed analysis of the molecular modifications at the shoot apex caused by the mutation. We show that meristem structure and function are not severely affected in the mutant. Major alterations, however, are observed at the periphery of the pin1 meristem, where organ initiation should occur. Although two very early markers of organ initiation, LEAFY and AINTEGUMENTA, are expressed at the periphery of the mutant meristem, the cells are not recruited into distinct primordia. Instead a ring-like domain expressing those primordium specific genes is observed around the meristem. This ring-like domain also expresses a boundary marker, CUP-SHAPED COTYLEDON 2, involved in organ separation, showing that the zone at the meristem periphery has a hybrid identity. This implies that PIN1 is not only involved in organ outgrowth, but that it is also necessary for organ separation and positioning. A model is presented in which PIN1 and the local distribution of auxin control phyllotaxis.

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The structure and function of centriolar rootlets

Journal of Cell Science ◽

10.1242/jcs.258544 ◽

2021 ◽

Vol 134 (16) ◽

Author(s):

Robert Mahen

Keyword(s):

Cellular Function ◽

Structure And Function ◽

Cellular Systems ◽

Macromolecular Assemblies ◽

Cellular Processes ◽

Holistic Understanding ◽

Eukaryotic Organisms ◽

And Function ◽

Knockout Animals

ABSTRACT To gain a holistic understanding of cellular function, we must understand not just the role of individual organelles, but also how multiple macromolecular assemblies function collectively. Centrioles produce fundamental cellular processes through their ability to organise cytoskeletal fibres. In addition to nucleating microtubules, centrioles form lesser-known polymers, termed rootlets. Rootlets were identified over a 100 years ago and have been documented morphologically since by electron microscopy in different eukaryotic organisms. Rootlet-knockout animals have been created in various systems, providing insight into their physiological functions. However, the precise structure and function of rootlets is still enigmatic. Here, I consider common themes of rootlet function and assembly across diverse cellular systems. I suggest that the capability of rootlets to form physical links from centrioles to other cellular structures is a general principle unifying their functions in diverse cells and serves as an example of how cellular function arises from collective organellar activity.

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Nemo-Like Kinase in Development and Diseases: Insights from Mouse Studies

International Journal of Molecular Sciences ◽

10.3390/ijms21239203 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9203

Author(s):

Renée Daams ◽

Ramin Massoumi

Keyword(s):

Tissue Homeostasis ◽

Spinocerebellar Ataxias ◽

Adult Tissue ◽

Signalling Molecules ◽

Cellular Processes ◽

Protein Map ◽

Mitogen Activated Protein ◽

And Function ◽

Deficient Mice

The Wnt signalling pathway is a central communication cascade between cells to orchestrate polarity and fate during development and adult tissue homeostasis in various organisms. This pathway can be regulated by different signalling molecules in several steps. One of the coordinators in this pathway is Nemo-like kinase (NLK), which is an atypical proline-directed serine/threonine mitogen-activated protein (MAP) kinase. Very recently, NLK was established as an essential regulator in different cellular processes and abnormal NLK expression was highlighted to affect the development and progression of various diseases. In this review, we focused on the recent discoveries by using NLK-deficient mice, which show a phenotype in the development and function of organs such as the lung, heart and skeleton. Furthermore, NLK could conduct the function and differentiation of cells from the immune system, in addition to regulating neurodegenerative diseases, such as Huntington’s disease and spinocerebellar ataxias. Overall, generations of NLK-deficient mice have taught us valuable lessons about the role of this kinase in certain diseases and development.

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TMEM231, mutated in orofaciodigital and Meckel syndromes, organizes the ciliary transition zone

The Journal of Cell Biology ◽

10.1083/jcb.201411087 ◽

2015 ◽

Vol 209 (1) ◽

pp. 129-142 ◽

Cited By ~ 59

Author(s):

Elle C. Roberson ◽

William E. Dowdle ◽

Aysegul Ozanturk ◽

Francesc R. Garcia-Gonzalo ◽

Chunmei Li ◽

...

Keyword(s):

Transition Zone ◽

Transmembrane Protein ◽

Syndrome Type ◽

Zone Formation ◽

Meckel Syndrome ◽

Evolutionarily Conserved ◽

Complex Functions ◽

And Function ◽

Type 3

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.

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C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0453 ◽

2017 ◽

Vol 28 (3) ◽

pp. 452-462 ◽

Cited By ~ 13

Author(s):

Madhavan Chalat ◽

Kody Moleschi ◽

Robert S. Molday

Keyword(s):

Endoplasmic Reticulum ◽

Atpase Activity ◽

Deletion Mutants ◽

Serine Residue ◽

Cellular Processes ◽

Phospholipid Asymmetry ◽

C Terminus ◽

And Function ◽

Phospholipid Flippase

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2.

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Lysosome Function in Cardiovascular Diseases

Cellular Physiology and Biochemistry ◽

10.33594/000000373 ◽

2021 ◽

Vol 55 (3) ◽

pp. 277-300

Keyword(s):

Intracellular Signaling ◽

Hydrolytic Enzymes ◽

Vascular Diseases ◽

Sphingolipid Metabolism ◽

Cellular Behavior ◽

Cellular Processes ◽

Vascular Regulation ◽

Large Numbers ◽

And Function

The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.

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