Improved mitochondrial function in the hearts of sarcolipin-deficient dystrophin and utrophin double-knockout mice.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin double-knockout (mdx:utrn-/-) mice in a sarcolipin (SLN) heterozygous-knockout (sln+/-) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn-/- mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes showed that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiomyocytes. These findings indicate that SLN upregulation plays a substantial role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD cardiomyopathy.

New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.

Recognition and remodeling of endosomal zones by sorting nexins.

The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3. Both proteins form sorting machines by recognizing membrane zones enriched in phosphatidylinositol 3-phosphate (PI3P), phosphatidylserine (PS) and cholesterol. This co-localized combination forms a unique "lipid codon" or lipidon that we propose is responsible for endosomal targeting, as revealed by structures and interactions of their PX domain-based readers. We outline a membrane recognition and remodeling mechanism for Snx1 and Snx3 involving this code element alongside transmembrane pH gradients, dipole moment-guided docking and specific protein-protein interactions. This generates an initial membrane-protein assembly (memtein) that then recruits retromer and additional PX proteins to recruit cell surface receptors for sorting to the trans-Golgi network (TGN), lysosome and plasma membranes. Post-translational modification (PTM) networks appear to regulate how the sorting machines form and operate at each level. The commonalities and differences between these sorting nexins show how the proteolipid code orchestrates parallel flows of molecular information from ribosome emergence to organelle genesis, and illuminates a universally applicable model of the membrane.

Research advances of MAL family members in tumorigenesis and tumor progression (Review).

The myelin and lymphocyte protein (MAL) family is a novel gene family first identified and characterized in 2002. This family is comprised of seven members, including MAL, MAL2, plasmolipin, MALL, myeloid differentiation­associated marker (MYADM), MYADML2 and CMTM8, which are located on different chromosomes. In addition to exhibiting extensive activity during transcytosis, the MAL family plays a vital role in the neurological, digestive, respiratory, genitourinary and other physiological systems. Furthermore, the intimate association between MAL and the pathogenesis, progression and metastasis of malignancies, attributable to several mechanisms such as DNA methylation has also been elucidated. In the present review, an overview of the structural and functional properties of the MAL family and the latest research findings regarding the relationship between several MAL members and various cancers is provided. Furthermore, the potential clinical and scientific significance of MAL is discussed and directions for future research are summarized.

Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila.

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.

Membranes are functionalized by a proteolipid code.

Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins.

Sarcolipin relates to fattening, but not sarco/endoplasmic reticulum Ca2+-ATPase uncoupling, in captive migratory gray catbirds.

In order to complete their energetically demanding journeys, migratory birds undergo a suite of physiological changes to prepare for long-duration endurance flight, including hyperphagia, fat deposition, reliance on fat as a fuel source, and flight muscle hypertrophy. In mammalian muscle, SLN is a small regulatory protein which binds to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and uncouples Ca2+ transport from ATP hydrolysis, increasing energy consumption, heat production, and cytosolic Ca2+ transients that signal for mitochondrial biogenesis, fatigue resistance and a shift to fatty acid oxidation. Using a photoperiod manipulation of captive gray catbirds (Dumetella carolinensis), we investigated whether SLN may play a role in coordinating the development of the migratory phenotype. In response to long-day photostimulation, catbirds demonstrated migratory restlessness and significant body fat stores, alongside higher SLN transcription while SERCA2 remained constant. SLN transcription was strongly correlated with h-FABP and PGC1α transcription, as well as fat mass. However, SLN was not significantly correlated with HOAD or CD36 transcripts or measurements of SERCA activity, SR membrane Ca2+ leak, Ca2+ uptake rates, pumping efficiency or mitochondrial biogenesis. Therefore, SLN may be involved in the process of storing fat and shifting to fat as a fuel, but the mechanism of its involvement remains unclear.

Reduced Proteolipid Protein 2 promotes endoplasmic reticulum stress-related apoptosis and increases drug sensitivity in acute myeloid leukemia.

BACKGROUND: The Proteolipid Protein 2 (PLP2), a protein in the Endoplasmic Reticulum (ER) membrane, has been reported to be highly expressed in various tumors. Previous studies have demonstrated that the reduced PLP2 can induce apoptosis and autophagy through ER stress-related pathways, leading to a decreased proliferation and aggressiveness. However, there is no research literature on the role of PLP2 in Acute Myeloid Leukemia (AML). METHODS: PLP2 expression, clinical data, genetic mutations, and karyotype changes from GEO, TCGA, and timer2.0 databases were analyzed through the R packages. The possible functions and pathways of cells were explored through GO, KEGG, and GSEA enrichment analysis using the clusterProfiler R package. Immuno-infiltration analysis was conducted using the Cibersort algorithm and the Xcell R package. RT-PCR and western blot techniques were employed to identify the PLP2 expression, examine the knockdown effects in THP-1 cells, and assess the expression of genes associated with endoplasmic reticulum stress and apoptosis. Flow cytometry was utilized to determine the apoptosis and survival rates of different groups. RESULTS: PLP2 expression was observed in different subsets of AML and other cancers. Enrichment analyses revealed that PLP2 was involved in various tumor-related biological processes, primarily apoptosis and lysosomal functions. Additionally, PLP2 expression showed a strong association with immune cell infiltration, particularly monocytes. In vitro, the knockdown of PLP2 enhanced endoplasmic reticulum stress-related apoptosis and increased drug sensitivity in THP-1 cells. CONCLUSIONS: PLP2 could be a novel therapeutic target in AML, in addition, PLP2 is a potential endoplasmic reticulum stress regulatory gene in AML.

Hypermetabolism in mice carrying a near-complete human chromosome 21.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Proteolipid protein 1 is involved in the regulation of intestinal motility and barrier function in the mouse.

Proteolipid protein 1 (Plp1) is highly expressed in enteric glia, labeling cells throughout the mucosa, muscularis, and the extrinsic innervation. Plp1 is a major constituent of myelin in the central and peripheral nervous systems, but the absence of myelin in the enteric nervous system (ENS) suggests another role for Plp1 in the gut. Although the functions of enteric glia are still being established, there is strong evidence that they regulate intestinal motility and permeability. To interrogate the role of Plp1 in enteric glia, we investigated gut motility, secretomotor function and permeability, and evaluated the ENS in mice lacking Plp1. We studied two time points: ∼3 mo (young) and >1 yr (old). Old Plp1 null mice exhibited increased fecal output, decreased fecal water content, faster whole gut transit times, reduced intestinal permeability, and faster colonic migrating motor complexes. Interestingly, in both young and old mice, the ENS exhibited normal glial and neuronal numbers as well as glial arborization density in the absence of Plp1. As Plp1-associated functions involve mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mapk/Erk1/2) signaling and Mapk/Erk1/2 are reported to have a regulatory role in intestinal motility, we measured protein expression of Erk1/2 and its active form in the small intestine. Old Plp1 null mice had reduced levels of phosphorylated-Erk1/2. Although Plp1 is not required for the normal appearance of enteric glial cells, it has a regulatory role in intestinal motility and barrier function. Our results suggest that functional changes mediated by Plp1-expressing enteric glia may involve Erk1/2 activation.NEW & NOTEWORTHY Here, we describe that Plp1 regulates gut motility and barrier function. The functional effects of Plp1 eradication are only seen in old mice, not young. The effects of Plp1 appear to be mediated through the Erk1/2 pathway.

Genomic Variant in NK-Lysin Gene Is Associated with T Lymphocyte Subpopulations in Pigs.

As an antimicrobial peptide, NK-lysin (NKL) plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White pigs and 74 Songliao Black pigs) were used to further explore the function of NLK gene in porcine immune system. The quantitative real-time PCR analysis detected the NKL gene's expression, and the result demonstrated that NKL mRNA was expressed in lung, spleen, stomach, kidney, liver and heart, and the expression level decreased sequentially. A single-nucleotide polymorphism (SNP, g.59070355 G > A) in intron 3 of the NKL gene was detected by PCR amplification and sequencing. The results of the Chi-square (χ2) test showed that the genotype of the SNP was consistent with the Hardy-Weinberg equilibrium. What's more, association analysis results showed the SNP in NKL gene was significantly associated with T lymphocyte subpopulations. Different genotypes had significant effects on the proportion of CD4-CD8-, CD4-CD8+, CD4+CD8+, CD8+, CD4+/CD8+ in peripheral blood (p < 0.05). These results further suggested that NKL could be recognized as a promising immune gene for swine disease resistance breeding.

The effects of neurogranin knockdown on SERCA pump efficiency in soleus muscles of female mice fed a high fat diet.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for the transport of Ca2+ from the cytosol into the sarcoplasmic reticulum at the expense of ATP, making it a regulator of both muscle relaxation and muscle-based energy expenditure. Neurogranin (Ng) is a small protein that negatively regulates calcineurin signaling. Calcineurin is Ca2+/calmodulin dependent phosphatase that promotes the oxidative fibre type in skeletal muscle and regulates muscle-based energy expenditure. A recent study has shown that calcineurin activation reduces SERCA Ca2+ transport efficiency, ultimately raising energy expenditure. Since the biomedical view of obesity states that it arises as an imbalance between energy intake and expenditure which favors the former, we questioned whether heterozygous Ng deletion (Ng+/- ) would reduce SERCA efficiency and increase energy expenditure in female mice fed a high-fat diet (HFD). Young (3-4-month-old) female wild type (WT) and Ng+/- mice were fed a HFD for 12 weeks with their metabolic profile being analyzed using metabolic cages and DXA scanning, while soleus SERCA efficiency was measured using SERCA specific Ca2+ uptake and ATPase activity assays. Ng+/- mice showed significantly less cage ambulation compared to WT mice but this did not lead to any added weight gain nor changes in daily energy expenditure, glucose or insulin tolerance despite a similar level of food intake. Furthermore, we observed significant reductions in SERCA's apparent coupling ratio which were associated with significant reductions in SERCA1 and phospholamban content. Thus, our results show that Ng regulates SERCA pump efficiency, and future studies should further investigate the potential cellular mechanisms.

Generation of Atrial-Specific Construct Using Sarcolipin Promoter-Associated CRM4 Enhancer.

Cardiac gene therapy has been hampered by off-target expression of gene of interest irrespective of variety of delivery methods. To overcome this issue, cardiac-specific promoters provide target tissue specificity, although expression is often debilitated compared to that of ubiquitous promoters. We have previously shown that sarcolipin promoter with an enhancer calsequestrin cis-regulatory module 4 (CRM4) combination has an improved atrial specificity. Moreover, it showed a minimal extra-atrial expression, which is a significant advantage for AAV9-mediated cardiac gene therapy. Therefore, it can be a useful tool to study and treat atrial-specific diseases such as atrial fibrillation. In this chapter, we introduce practical and simple methodology for atrial-specific gene therapy using sarcolipin promoter with an enhancer CRM4.

Single Vesicle Fluorescence-Bleaching Assay for Multi-Parameter Analysis of Proteoliposomes by Total Internal Reflection Fluorescence Microscopy.

Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analysis of multiple parameters (physical size, tightness, unilamellarity, membrane protein content, and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup.

Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020).

Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells.

BACKGROUND: Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat liberation. Exploiting this thermogenic mechanism in skeletal muscle may provide an attractive strategy to counteract obesity and associated metabolic disorders. In the present study, we have investigated the role of SLN on substrate metabolism in human skeletal muscle cells. METHODS AND RESULTS: After generation of skeletal muscle cells with stable SLN knockdown (SLN-KD), cell viability, glucose and oleic acid (OA) metabolism, mitochondrial function, as well as gene expressions were determined. Depletion of SLN did not influence cell viability. However, glucose and OA oxidation were diminished in SLN-KD cells compared to control myotubes. Basal respiration measured by respirometry was also observed to be reduced in cells with SLN-KD. The metabolic perturbation in SLN-KD cells was reflected by reduced gene expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and forkhead box O1 (FOXO1). Furthermore, accumulation of OA was increased in cells with SLN-KD compared to control cells. These effects were accompanied by increased lipid formation and incorporation of OA into complex lipids. Additionally, formation of complex lipids and free fatty acid from de novo lipogenesis with acetate as substrate was enhanced in SLN-KD cells. Detection of lipid droplets using Oil red O staining also showed increased lipid accumulation in SLN-KD cells. CONCLUSIONS: Overall, our study sheds light on the importance of SLN in maintaining metabolic homeostasis in human skeletal muscle. Findings from the current study suggest that therapeutic strategies involving SLN-mediated futile cycling of SERCA might have significant implications in the treatment of obesity and associated metabolic disorders.

PLP2-derived peptide Rb4 triggers PARP-1-mediated necrotic death in murine melanoma cells.

Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.

Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2.

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.