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Biochemistry Jul 2022Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca-ATPase (SERCA), which...
Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.
Topics: Animals; Calcium; Calcium Signaling; Calcium-Binding Proteins; Humans; Mammals; Molecular Dynamics Simulation; Muscle Proteins; Peptides; Proteolipids; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases
PubMed: 35771007
DOI: 10.1021/acs.biochem.2c00246 -
Communications Biology Jun 2021It is well-established that the secondary active transporters Glt and Glt catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic...
It is well-established that the secondary active transporters Glt and Glt catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified Glt, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on Glt using this equation allowed for determination of the turnover number (0.14 s), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented Glt proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.
Topics: Aspartic Acid; Biological Transport; Excitatory Amino Acid Transporter 3; Proteolipids; Pyrococcus horikoshii; Sodium; Thermococcus
PubMed: 34140623
DOI: 10.1038/s42003-021-02267-y -
Nature Structural & Molecular Biology Feb 2022The Na/H exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na/Li exchanger linked to the pathogenesis of diabetes mellitus and essential...
The Na/H exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na/Li exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na/H exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.
Topics: Amino Acid Sequence; Animals; Antiporters; Binding Sites; Bison; Cryoelectron Microscopy; Humans; Lipid Metabolism; Mass Spectrometry; Models, Molecular; Molecular Dynamics Simulation; Nanostructures; Protein Multimerization; Proteolipids; Sodium-Hydrogen Exchangers; Static Electricity
PubMed: 35173351
DOI: 10.1038/s41594-022-00738-2 -
Orphanet Journal of Rare Diseases Mar 2022The natural history and genotype-phenotype correlation of Pelizaeus-Merzbacher disease (PMD) of Chinese patients has been rarely reported.
BACKGROUND
The natural history and genotype-phenotype correlation of Pelizaeus-Merzbacher disease (PMD) of Chinese patients has been rarely reported.
METHOD
Patients who met the criteria for PMD were enrolled in our study. Genomic analysis was conducted by multiplex ligation probe amplification (MLPA) and Sanger or whole-exome sequencing (WES). Natural history differences and genotype-phenotype correlations were analyzed.
RESULT
A total of 111 patients were enrolled in our follow-up study. The median follow-up interval was 53 m (1185). Among PMD patients, developmental delay was the most common sign, and nystagmus and hypotonia were the most common initial symptoms observed. A total of 78.4% of the patients were able to control their head, and 72.1% could speak words. However, few of the patients could stand (9.0%) or walk (4.5%) by themselves. Nystagmus improved in more than half of the patients, and hypotonia sometimes deteriorated to movement disorders. More PLP1 point mutations patients were categorized into severe group, while more patients with PLP1 duplications were categorized into mild group (p < 0.001). Compared to patients in mild groups, those in the severe group had earlier disease onset and had acquired fewer skills at a later age.
CONCLUSION
PMD patients have early disease onset with nystagmus and hypotonia followed by decreased nystagmus and movement disorders, such as spasticit. Patients with PLP1 duplication were more likely to be categorized into the mild group, whereas patients with point mutations were more likely to be categorized into the severe group.
Topics: China; Follow-Up Studies; Genetic Association Studies; Humans; Myelin Proteolipid Protein; Pelizaeus-Merzbacher Disease
PubMed: 35346287
DOI: 10.1186/s13023-022-02267-z -
Scientific Reports Jan 2018Keratoconus is a common degenerative corneal disease that can lead to significant visual morbidity, and both genetic and environmental factors have been implicated in... (Comparative Study)
Comparative Study
Keratoconus is a common degenerative corneal disease that can lead to significant visual morbidity, and both genetic and environmental factors have been implicated in its pathogenesis. We compared the transcriptome of keratoconus and control epithelium using RNA-Seq. Epithelial tissues were obtained prior to surgery from keratoconus and myopia control patients, undergoing collagen cross-linking and photorefractive keratectomy, respectively. We identified major differences in keratoconus linked to cell-cell communication, cell signalling and cellular metabolism. The genes associated with the Hedgehog, Wnt and Notch1 signaling pathways were down-regulated in keratoconus. We also identified plasmolipin and Notch1 as being significantly reduced in keratoconus for both gene and protein expression (p < 0.05). Plasmolipin is a novel protein identified in human corneal epithelium, and has been demonstrated to have a key role in epithelial cell differentiation in other tissues. This study shows altered gene and protein expression of these three proteins in keratoconus, and further studies are clearly warranted to confirm the functional role of these proteins in the pathogenesis of keratoconus.
Topics: Adolescent; Adult; Epithelium, Corneal; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Humans; Keratoconus; Male; Middle Aged; Myelin and Lymphocyte-Associated Proteolipid Proteins; Myopia; Receptor, Notch1; Sequence Analysis, RNA; Wnt Signaling Pathway; Young Adult
PubMed: 29321650
DOI: 10.1038/s41598-017-18480-x -
Nature Communications Apr 2021Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized...
Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.
Topics: Cell Membrane; Copper-Transporting ATPases; Detergents; Escherichia coli Proteins; Exoskeleton Device; Immobilization; Kinetics; Lipid Bilayers; Membrane Proteins; Micelles; Phospholipids; Proteolipids; Scattering, Radiation; Unilamellar Liposomes; X-Ray Diffraction
PubMed: 33850135
DOI: 10.1038/s41467-021-22285-y -
Annals of Clinical and Translational... Mar 2023Spastic paraplegia type 2 (SPG2) is an X-linked recessive (XLR) form of hereditary spastic paraplegia (HSP) caused by mutations in proteolipid protein 1 (PLP1) gene. We...
OBJECTIVE
Spastic paraplegia type 2 (SPG2) is an X-linked recessive (XLR) form of hereditary spastic paraplegia (HSP) caused by mutations in proteolipid protein 1 (PLP1) gene. We described the clinical and genetic features of three unrelated families with PLP1 mutations and reviewed PLP1-related cases worldwide to summarize the genotype-phenotype correlations.
METHODS
The three probands were 23, 26, and 27 years old, respectively, with progressively aggravated walking difficulty as well as lower limb spasticity. Detailed physical examination showed elevated muscle tone, hyperreflexia, and Babinski signs in lower limbs. Brain MRI examinations were investigated for all cases. PLP1 mutations were identified by whole exome sequencing, followed by Sanger sequencing, family co-segregation, and phenotypic reevaluation.
RESULTS
A total of eight patients with SPG2 were identified in these three families. The probands additionally had cognitive impairment, urinary or fecal incontinence, ataxia, and white matter lesions (WML) in periventricular regions, with or without kinetic tremor. Three hemizygous mutations in PLP1 were identified, including c.453+159G>A, c.834A>T (p.*278C), and c.434G>A (p.W145*), of which c.834A>T was first associated with HSP.
INTERPRETATION
We identified three families with complicated SPG2 due to three PLP1 mutations. Our study supports the clinically inter-and intra-family heterogeneity of SPG2. The periventricular region WML and cognitive impairment are the most common characteristics. The kinetic tremor in upper limbs was observed in 2/3 families, suggesting the spectrum of PLP1-related disorders is still expanding.
Topics: Humans; Spastic Paraplegia, Hereditary; Myelin Proteolipid Protein; Tremor; Mutation
PubMed: 36622199
DOI: 10.1002/acn3.51722 -
Oncotarget Apr 2016The aim of the present study was to determine whether MALL expression is associated with colon cancer progression and patient survival. MALL mRNA expression was reduced...
The aim of the present study was to determine whether MALL expression is associated with colon cancer progression and patient survival. MALL mRNA expression was reduced in the tumor tissues of 70% of the colon cancer patients and 75% of the rectal cancer patients as compared to their normal tissues. MALL protein was also significantly reduced in the tumor tissues of colon cancer patients (P < 0.001). Increased LOH and methylation of MALL was observed in tumor tissues as compared to normal tissues. Reduced MALL expression was associated with vessin invasion, disease recurrence and metastasis or death (P ≤ 0.027). Furthermore, patients with MALL-negative tumors had significantly decreased overall survival (OS) and disease-free survival (DFS) (P < 0.008 and P < 0.011, respectively). Univariate analysis indicated that MALL expression was significantly associated with OS and DFS. Finally, overexpression of MALL suppressed HCT116 and SW480 cell proliferation and inhibited HCT116 migration. MALL may play a role in colorectal cancer progression as suppression of its expression in tumor tissues negatively impacts colorectal cancer patient survival. Further analyses are required to determine if reduced MALL expression is due to LOH and/or methylation.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Myelin and Lymphocyte-Associated Proteolipid Proteins; Prognosis; Proportional Hazards Models; Young Adult
PubMed: 26992238
DOI: 10.18632/oncotarget.8094 -
American Journal of Physiology. Cell... Oct 2019Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca-ATPase (SERCA), ameliorates severe muscular dystrophy in mice....
Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in CC resulted in decreased SERCA pump activity, reduced SR Ca load, and increased intracellular Ca () concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.
Topics: Animals; Calcium; Cell Differentiation; Dogs; Dystrophin; Mice, Knockout; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Myoblasts; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases
PubMed: 31365291
DOI: 10.1152/ajpcell.00146.2019 -
The Journal of Biological Chemistry Jun 2020The formation of a mature, multilayered myelin sheath requires the compaction of lipid bilayers, but the molecular mechanism by which these bilayers condense is an open...
The formation of a mature, multilayered myelin sheath requires the compaction of lipid bilayers, but the molecular mechanism by which these bilayers condense is an open question. In this issue, Ruskamo find that peripheral myelin protein P2 forms an ordered three-dimensional lattice within model membranes using polar lipid liposomes. These data will help to understand the assembly, function, and structure of the myelin sheath.
Topics: Cryoelectron Microscopy; Lipid Bilayers; Myelin Proteins; Myelin Sheath; Proteolipids; X-Ray Diffraction
PubMed: 32591442
DOI: 10.1074/jbc.H120.014273