Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits

The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the “regulins”. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA—formed by transmembrane helices M2, M6, and M9—can result in distinct functional outcomes.

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Upregulation of the Sarco-Endoplasmic Reticulum Calcium ATPase 1 Truncated Isoform Plays a Pathogenic Role in Alzheimer’s Disease

Cells ◽

10.3390/cells8121539 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1539 ◽

Cited By ~ 4

Author(s):

Bussiere ◽

Oulès ◽

Mary ◽

Vaillant-Beuchot ◽

Martin ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Neuroblastoma Cells ◽

Amyloid Β ◽

Aβ Oligomers ◽

Human Neuroblastoma ◽

Endoplasmic Reticulum Calcium ◽

Protein Levels ◽

Functional Consequences ◽

Β Amyloid

Dysregulation of the Endoplasmic Reticulum (ER) Ca2+ homeostasis and subsequent ER stress activation occur in Alzheimer Disease (AD). We studied the contribution of the human truncated isoform of the sarco-endoplasmic reticulum Ca2+ ATPase 1 (S1T) to AD. We examined S1T expression in human AD-affected brains and its functional consequences in cellular and transgenic mice AD models. S1T expression is increased in sporadic AD brains and correlates with amyloid β (Aβ) and ER stress chaperone protein levels. Increased S1T expression was also observed in human neuroblastoma cells expressing Swedish-mutated β-amyloid precursor protein (βAPP) or treated with Aβ oligomers. Lentiviral overexpression of S1T enhances in return the production of APP C-terminal fragments and Aβ through specific increases of β-secretase expression and activity, and triggers neuroinflammation. We describe a molecular interplay between S1T-dependent ER Ca2+ leak, ER stress and βAPP-derived fragments that could contribute to AD setting and/or progression.

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Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

10.1101/2020.10.22.349522 ◽

2020 ◽

Author(s):

Valeria Marquez-Miranda ◽

Maximiliano Rojas ◽

Yorley Duarte ◽

Ignacio Diaz-Franulic ◽

Miguel Holmgren ◽

...

Keyword(s):

Transmembrane Domain ◽

Lung Epithelial Cells ◽

Transmembrane Helices ◽

Cellular Processes ◽

Charged Amino Acids ◽

Coordination Sites ◽

Lung Epithelial ◽

Dynamics Simulations ◽

Positively Charged

AbstractSARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer’s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain’s potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity’s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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Sarcolambans are phospholamban- and sarcolipin-like regulators of the sarcoplasmic reticulum calcium pump SERCA

10.1101/2020.07.19.211243 ◽

2020 ◽

Author(s):

Jessi J. Bak ◽

Rodrigo Aguayo-Ortiz ◽

Muhammad Bashir Khan ◽

Seth L. Robia ◽

M. Joanne Lemieux ◽

...

Keyword(s):

Skeletal Muscle ◽

Maximal Activity ◽

Cytosolic Calcium ◽

Protein Docking ◽

Calcium Pump ◽

Evolutionary Divergence ◽

Mammalian Skeletal Muscle ◽

Regulatory Subunits ◽

Endoplasmic Reticulum Calcium ◽

Dynamics Simulations

ABSTRACTFrom insects to humans, calcium signaling is essential for life. An important part of this process is the sarco-endoplasmic reticulum calcium pump SERCA, which maintains low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, this is a tightly controlled system where SERCA interacts with tissuespecific regulatory subunits such as phospholamban in cardiac muscle and sarcolipin in skeletal muscle. With the recent discovery of the sarcolambans, the family of calcium pump regulatory subunits also appears to be ancient, spanning more than 550 million years of evolutionary divergence from insects to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and range from vaguely phospholamban-like to vaguely sarcolipin-like. Here, our goal was to investigate select sarcolamban peptides for their ability to regulate calcium pump activity. For a side-by-side comparison of diverse sarcolamban peptides, we tested them against mammalian skeletal muscle SERCA1a. This allowed us to determine if the sarcolamban peptides mimic phospholamban and sarcolipin in their regulatory activities. Four sarcolamban peptides were chosen from different invertebrate species. Of these, we were able to express and purify sarcolamban peptides from bumble bee, water flea, and tadpole shrimp. Sarcolamban peptides were co-reconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. While all peptides were super-inhibitors of SERCA, they exhibited either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations were used to reveal novel features of insect versus mammalian calcium pumps and the sarcolamban regulatory subunits.

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Faculty Opinions recommendation of Recognition of transmembrane helices by the endoplasmic reticulum translocon.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023628.287994 ◽

2005 ◽

Author(s):

Tracy Palmer

Keyword(s):

Endoplasmic Reticulum ◽

Transmembrane Helices

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Familial hemiplegic migraine type 2 due to a novel missense mutation in ATP1A2

The Journal of Headache and Pain ◽

10.1186/s10194-021-01221-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Fabio Antonaci ◽

Sabrina Ravaglia ◽

Gaetano S. Grieco ◽

Stella Gagliardi ◽

Cristina Cereda ◽

...

Keyword(s):

Case Reports ◽

3D Structure ◽

Familial Hemiplegic Migraine ◽

Transmembrane Helices ◽

Hemiplegic Migraine ◽

Case Presentation ◽

Italian Family ◽

Protein Prediction ◽

Functional Consequences

Abstract Background The mechanisms of genotype-phenotype interaction in Familiar Hemiplegic migraine type 2 (FHM2) are still far from clear. Different ATP1A2 mutations have been described, with a spectrum of phenotypes ranging from mild to severe. No genotype-phenotype correlations have been attempted. Case presentation We describe an Italian family with FHM and a missense ATP1A2 variant (L425H) not previously described. The clinical picture was mild in all the affected members. Conclusions Co-segregation of the variant with the aura phenotype was complete in this family, suggesting a 100% penetrance. In silico protein prediction softwares indicate that this variant may change the 3D structure of ATPA1A2 at the cytoplasmic loop between the two central transmembrane helices. Milder FHM phenotypes are rarely reported in literature, likely because case reports are biased towards the most severe phenotypes, with milder forms possibly misdiagnosed as sporadic migraine with aura forms (MAs), even with complex auras. Further studies taking into account intra-familiar variability and functional consequences on the channel protein may help clarify genotype-phenotype correlations.

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The role of endoplasmic reticulum calcium pumps during cytosolic calcium spiking in pancreatic acinar cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)41521-9 ◽

1993 ◽

Vol 268 (30) ◽

pp. 22262-22264

Author(s):

C.C. Petersen ◽

O.H. Petersen ◽

M.J. Berridge

Keyword(s):

Endoplasmic Reticulum ◽

Acinar Cells ◽

Cytosolic Calcium ◽

Pancreatic Acinar Cells ◽

Endoplasmic Reticulum Calcium ◽

Calcium Pumps ◽

Calcium Spiking

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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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Computational Design of Macrocyclic Binders of S100B(ββ): Novel Peptide Theranostics

Molecules ◽

10.3390/molecules26030721 ◽

2021 ◽

Vol 26 (3) ◽

pp. 721

Author(s):

Srinivasaraghavan Kannan ◽

Pietro G. A. Aronica ◽

Thanh Binh Nguyen ◽

Jianguo Li ◽

Chandra S. Verma

Keyword(s):

Molecular Dynamics ◽

Computational Design ◽

Diagnostic Tools ◽

Stapled Peptides ◽

Altered Expression ◽

Cellular Processes ◽

High Affinity ◽

Limited Success ◽

Dynamics Simulations ◽

Partner Proteins

S100B(ββ) proteins are a family of multifunctional proteins that are present in several tissues and regulate a wide variety of cellular processes. Their altered expression levels have been associated with several human diseases, such as cancer, inflammatory disorders and neurodegenerative conditions, and hence are of interest as a therapeutic target and a biomarker. Small molecule inhibitors of S100B(ββ) have achieved limited success. Guided by the wealth of available experimental structures of S100B(ββ) in complex with diverse peptides from various protein interacting partners, we combine comparative structural analysis and molecular dynamics simulations to design a series of peptides and their analogues (stapled) as S100B(ββ) binders. The stapled peptides were subject to in silico mutagenesis experiments, resulting in optimized analogues that are predicted to bind to S100B(ββ) with high affinity, and were also modified with imaging agents to serve as diagnostic tools. These stapled peptides can serve as theranostics, which can be used to not only diagnose the levels of S100B(ββ) but also to disrupt the interactions of S100B(ββ) with partner proteins which drive disease progression, thus serving as novel therapeutics.

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