Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

A novel machine learning-based screening identifies statins as inhibitors of the calcium pump SERCA.

We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.

Setting the Record Straight Regarding Tools That Assess Risk for Youth with Sexually Abusive Behavior.

This critique alerts practicing professionals of the multiple misleading statements in the recently published article entitled, "A compendium of risk and needs tools for assessing male youths at-risk to and/or who have engaged in sexually abusive behaviors." This critique corrects the erroneous information contained in Jung and Thomas' article, providing current accurate information related to the important distinct differences of available standardized risk assessment tools used in forensic settings with youths who have engaged in sexually abusive behaviors. Erroneous statements by other researchers and authors in the field are also discussed. Forensic cases are distinctively different from others seen in clinical settings, requiring specific knowledge and skill set, a notable distinction not often mentioned in research literature.

Functional Role of an Unusual Transmembrane Acidic Residue in the Calcium Pump Regulator Myoregulin.

Myoregulin (MLN) is a member of the regulin family, a group of homologous membrane proteins that bind to and regulate the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). MLN, which is expressed in skeletal muscle, contains an acidic residue in its transmembrane domain. The location of this residue, Asp35, is unusual because the relative occurrence of aspartate is very rare (<0.2%) within the transmembrane helix regions. Therefore, we used atomistic simulations and ATPase activity assays of protein co-reconstitutions to probe the functional role of MLN residue Asp35. These structural and functional studies showed Asp35 has no effects on SERCA's affinity for Ca2+ or the structural integrity of MLN in the lipid bilayer. Instead, Asp35 controls SERCA inhibition by populating a bound-like orientation of MLN. We propose Asp35 provides a functional advantage over other members of the regulin family by populating preexisting MLN conformations required for MLN-specific regulation of SERCA. Overall, this study provides new clues about the evolution and functional divergence of the regulin family and offers novel insights into the functional role of acidic residues in transmembrane protein domains.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Fermented food: Should patients with cardiometabolic diseases go back to an early neolithic diet?

Fermentation has been used since the Early Neolithic period to preserve foods. It has inherent organoleptic and nutritive properties that bestow health benefits, including reducing inflammation and oxidative stress, supporting the growth of salutogenic microbiota, enhancing intestinal mucosal protection and promoting beneficial immunometabolic health effects. The fermentation of food with specific microbiota increases the production salutogenic bioactive compounds that can activate Nrf2 mediated cytoprotective responses and mitigate the effects of the 'diseasome of aging' and its associated inflammageing, which presents as a prominent feature of obesity, type-2 diabetes, cardiovascular and chronic kidney disease. This review discusses the importance of fermented food in improving health span, with special reference to cardiometabolic diseases.

We are Not Adult Convicted Sex Offenders; We are Your Youth! the Call for Developmentally-Gender Sensitive Assessment of Youth Who are Sexually Abusive.

This article renews the call by Chaffin and Bonner in 1998 to cease treating youth who engage in sexually abusive behaviors like adult male convicted sex offenders. The author urges that professionals cease imposing an adult-centered convicted sex offender paradigm in assessment and treatment. A new paradigm is advocated that considers sexual developmental and gender issues, along with judicial status in all areas of intervention (i.e., clinical and risk assessment, treatment/service planning, and possible treatment [when needed]). The paradigm is grounded on the Family Lovemap model, a conceptual framework for assessing the youth's bio-physio-psycho-social-sexual and erotic development. Discussed is the evidence-based, developmentally and gender sensitive risk-level tool, MEGA♪ as a viable option for assessing risk for persistent coarse sexual improprieties and sexually abusive behaviors in youth ages 4-19 (adjudicated and non-adjudicated). Selected significant empirical findings are presented from 3,901 youths who were participants in samples of the MEGA♪ validation studies.

The Unpopular Victims: Individuals Who Are Sexually Abusive - The Black Swans.

This article contends that youth who engage in persistent coarse sexual improprieties and/or, sexually abusive behaviors are too often viewed primarily through a criminological lens, regardless of their age, gender, judicial status, and/or experiences of past trauma/victimization. Such a posture likely falsifies the clinical perception of the individual referred for "treatment." Assessment and interventions for these youth must be holistic and idiosyncratic, considering numerous multiplex developmental variables (i.e., overall human sexual development, gender identity, sexual identity, sexual orientation, erotic development, intimacy deficits, adverse childhood experiences/trauma), as well as sociological and anthropological fundamentals. The lens for assessing and intervening with youth must also include in its focus the constantly changing Zeitgeist, that is, the spirit or the mood of the times. The proposed lens is applicable to all youth, adjudicated and non-adjudicated. Specific considerations are discussed related to those youth with a history of significant child maltreatment who later engage in persistent coarse sexual improprieties and/or sexually abusive behaviors.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-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.

Maternofetal outcomes in early-onset gestational diabetes: does weight gain matter?

AIM: Women with early-onset gestational diabetes mellitus (GDM) have overall lower gestational weight gain (GWG) compared to those with later-onset GDM, albeit with usually worse maternofetal outcomes. We intent to investigate the association between inadequate GWG and maternofetal outcomes in pregnant women with early-onset GDM. METHODS: We performed a retrospective study of women with early-onset GDM based on the National Registry of GDM. Three study groups were defined according to the recommendations of the Institute of Medicine for GWG: excessive GWG (eGWG), adequate (aGWG) or insufficient (iGWG). RESULTS: A total of 8040 pregnant women were included: 27% (n = 2170) eGWG, 31% (n = 2492) aGWG and 42% (n = 3378) iGWG. Preeclampsia (4.3 vs 3 vs 1.6%, p < 0.001), polyhydramnios (3.1 vs 2.3 vs 1.8%, p = 0.008) and cesarean section (37.4 vs 34.1 vs 29.5%, p < 0.001) were significantly more frequent among women with eGWG. Additionally, there was a higher frequency of macrosomia (8.1 vs 3.6 vs 2.4%, p < 0.001), large-for-gestational-age (8.2 vs 3.7 vs 2.6%, p < 0.001) and birth trauma (2.6 vs 1.5 vs 1.1%, p < 0.001) in this group. On the other hand, fetal death (0.2 vs 0.2 vs 0.5%, p = 0.04), small-for-gestational-age (9 vs 10.3 vs 14.9, p < 0.001) and preterm delivery (5.6 vs 7.1 vs 7.5%, p = 0.03) were more frequent in iGWG group. CONCLUSIONS: Over two-thirds of pregnant women with early-onset GDM had inappropriate GWG, which was significantly associated with adverse maternofetal outcomes. Weight management must be a focus of special attention in women with early-onset GDM, beyond glycemic control, to achieve healthy pregnancy outcomes.

Genome-wide scan reveals genomic regions and candidate genes underlying direct and maternal effects of preweaning calf mortality in Nellore cattle.

We conducted analysis to estimate genetic parameters and to identify genomic regions and candidate genes affecting direct and maternal effects of preweaning calf mortality (PWM) in Nellore cattle. Phenotypic records of 67,196 animals, and 8443 genotypes for 410,936 SNPs were used. Analysis were performed through the weighted single-step GBLUP approach and considering a threshold animal model via Bayesian Inference. Direct and maternal heritability estimates were of 0.2143 ± 0.0348 and 0.0137 ± 0.0066, respectively. The top 10 genomic regions accounted for 13.61 and 14.23% of the direct and maternal additive genetic variances and harbored a total of 63 and 91 positional candidate genes, respectively. Two overlapping regions on BTA2 were identified for both direct and maternal effects. Candidate genes are involved in biological mechanisms i.e. embryogenesis, immune response, feto-maternal communication, circadian rhythm, hormone alterations, myometrium adaptation, and milk secretion, which are critical for the successful calf growth and survival during preweaning period.

Food consumption on campus is associated with meal eating patterns among college students.

This is a cross-sectional analysis of data obtained in the baseline of the Longitudinal Study on the Lifestyle and Health of University Students (n 685) carried out in a public Brazilian university. Food intake was assessed using a 24-h dietary recall. Dietary patterns (DP) for breakfast, lunch and dinner were identified using principal component analysis. Generalised linear models were used to analyse the variables associated with each DP. Three DP were extracted for each meal: breakfast: 'White bread and butter/margarine', 'Coffee and tea' and 'Sausages, whole wheat bread and cheese'; lunch: 'Traditional', 'Western' and 'Vegetarian' and dinner: 'Beans, rice and processed juice', 'White bread and butter/margarine' and 'White meat, eggs and natural juice'. Students who had meals at the campus showed greater adherence to the 'White bread and butter/margarine' (exp (ßadj) = 1·15, 95 % CI 1·11, 1·19) and 'Coffee and tea' (exp (ßadj) = 1·06, 95 % CI 1·02, 1·10) breakfast patterns; 'Western' lunch pattern (exp (ßadj) = 1·04, 95 % CI 1·01, 1·08) and to the 'Beans, rice and processed juice' dinner pattern (exp (ßadj) = 1·10, 95 % CI 1·06, 1·14). Having meals at the campus was associated with lower adherence to the 'Sausages, whole wheat bread and cheese' breakfast pattern (exp (ßadj) = 0·93, 95 % CI 0·89, 0·97), 'Traditional' lunch pattern (exp (ßadj) = 0·96, 95 % CI 0·93, 0·99) and to the 'White bread and butter/margarine' (exp (ßadj) = 0·96, 95 % CI 0·93, 0·99) and 'White meat, eggs and natural juice' (exp (ßadj) = 0·96, 95 % CI 0·93, 0·99) dinner pattern. The food environment at campus may influence students' DP. Recognising meal eating patterns is important to support healthy eating promotion strategies on campus. Adjustments in the University Canteen menu could contribute to healthier eating choices among students.

Structural Basis for the Function of the C-Terminal Proton Release Pathway in the Calcium Pump.

The calcium pump (sarco/endoplasmic reticulum Ca2+-ATPase, SERCA) plays a major role in calcium homeostasis in muscle cells by clearing cytosolic Ca2+ during muscle relaxation. Active Ca2+ transport by SERCA involves the structural transition from a low-Ca2+ affinity E2 state toward a high-Ca2+ affinity E1 state of the pump. This structural transition is accompanied by the countertransport of protons to stabilize the negative charge and maintain the structural integrity of the transport sites and partially compensate for the positive charges of the two Ca2+ ions passing through the membrane. X-ray crystallography studies have suggested that a hydrated pore located at the C-terminal domain of SERCA serves as a conduit for proton countertransport, but the existence and function of this pathway have not yet been fully characterized. We used atomistic simulations to demonstrate that in the protonated E2 state and the absence of initially bound water molecules, the C-terminal pore becomes hydrated in the nanosecond timescale. Hydration of the C-terminal pore is accompanied by the formation of water wires that connect the transport sites with the cytosol. Water wires are known as ubiquitous proton-transport devices in biological systems, thus supporting the notion that the C-terminal domain serves as a conduit for proton release. Additional simulations showed that the release of a single proton from the transport sites induces bending of transmembrane helix M5 and the interaction between residues Arg762 and Ser915. These structural changes create a physical barrier against full hydration of the pore and prevent the formation of hydrogen-bonded water wires once proton transport has occurred through this pore. Together, these findings support the notion that the C-terminal proton release pathway is a functional element of SERCA and also provide a mechanistic model for its operation in the catalytic cycle of the pump.

A Critical Assessment of the Youth Needs and Progress Scale.

Evaluated is a recently developed tool, the Youth Needs and Progress Scale (YNPS). Reported is the surprisingly palpable lack of adherence to scientific standards. A demonstrable absence of attention and accordance to scientific methods related to research and developing psychometrics is evident, that is, taking a colander approach when constructing the tool. The rudimentary steps of this project were described by Kang et al. (2019) and subsequently critiqued by this author. Significant concerns previously delineated were not addressed, but rather crystalized in a substantial U.S. federally tax funded grant for a substandard tool. These are described in this article (i.e., lack of adequate literature review, citing selective research findings, referencing face validity tools, problematic research design). The key concern is the reliance on risk recidivism tools (J-SOAP-II and ERASOR), partially based on adult research and empirically shown to have inconsistently performed in risk assessment studies.

Multiscale Simulation Reveals Passive Proton Transport Through SERCA on the Microsecond Timescale.

The sarcoplasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions from the cytoplasm to the reticulum lumen at the expense of ATP hydrolysis. In addition to transporting Ca2+, SERCA facilitates bidirectional proton transport across the sarcoplasmic reticulum to maintain the charge balance of the transport sites and to balance the charge deficit generated by the exchange of Ca2+. Previous studies have shown the existence of a transient water-filled pore in SERCA that connects the Ca2+ binding sites with the lumen, but the capacity of this pathway to sustain passive proton transport has remained unknown. In this study, we used the multiscale reactive molecular dynamics method and free energy sampling to quantify the free energy profile and timescale of the proton transport across this pathway while also explicitly accounting for the dynamically coupled hydration changes of the pore. We find that proton transport from the central binding site to the lumen has a microsecond timescale, revealing a novel passive cytoplasm-to-lumen proton flow beside the well-known inverse proton countertransport occurring in active Ca2+ transport. We propose that this proton transport mechanism is operational and serves as a functional conduit for passive proton transport across the sarcoplasmic reticulum.

Dynamics-Driven Allostery Underlies Ca2+-Mediated Release of SERCA Inhibition by Phospholamban.

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a control function that regulates cytosolic and SR Ca2+ levels. Although experimental and computational studies alone have led to a greater insight into SERCA-PLB regulation, the structural mechanisms for Ca2+ binding reversing inhibition of the complex remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 µs and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to binding of a single Ca2+ ion. Complementary MD simulations and FRET experiments showed that open-to-closed transitions in the structure of the headpiece underlie PLB inhibition of SERCA, and binding of a single Ca2+ ion is sufficient to shift the protein population toward a structurally closed structure of the complex. Closure is accompanied by functional interactions between the N-domain ß5-ß6 loop and the A-domain and the displacement of the catalytic phosphorylation domain toward a competent structure. We propose that reversal of SERCA-PLB inhibition is achieved by stringing together its controlling modules (A-domain and loop Nß5-ß6) with catalytic elements (P-domain) to regulate function during intracellular Ca2+ signaling. We conclude that binding of a single Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that relieve SERCA inhibition by PLB. Understanding allosteric regulation is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA.

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn4-Ala and Thr5-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.

Crotoxin down-modulates pro-inflammatory cells and alleviates pain on the MOG35-55-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a Central Nervous System inflammatory demyelinating disease that has as primary symptoms losses of sensory and motor functions, including chronic pain. To date, however, few studies have investigated the mechanisms of chronic pain in animal models of MS since locomotor impairments render difficult its evaluation. It was previously demonstrated that in the MOG35-55-induced EAE, an animal model of MS, the hypernociception appears before the onset of motor disability, allowing for the study of these two phenomena separately. Here, we evaluated the effect of crotoxin (CTX), a neurotoxin isolated from the Crotalus durissus terrificus snake venom that displays, at non-toxic dose, antinociceptive, anti-inflammatory and immunomodulatory effects, in the pain and in symptoms progression of EAE. The pain threshold of female C57BL/6 mice decreased at the 4th day after immunization, while the first sign of disease appeared around the 11st-12nd days, coinciding with the onset of motor abnormalities. CTX (40 µg/kg, s.c.) administered in a single dose on the 5th day after immunization, induced a long-lasting analgesic effect (5 days), without interfering with the clinical signs of the disease. On the other hand, when crotoxin was administered for 5 consecutive days, from 5th-9th day after immunization, it induced analgesia and also reduced EAE progression. The antinociceptive effect of crotoxin was blocked by Boc-2 (0.5 mg/kg, i.p.), a selective antagonist of formyl peptide receptors, by NDGA (30 µg/kg, i.p.), a lipoxygenase inhibitor and by atropine sulfate (10 mg/kg, i.p.), an antagonist of muscarinic receptors, administered 30 min before CTX. CTX was also effective in decreasing EAE clinical signs even when administered after its onset. Regarding the interactions between neurons and immunocompetent cells, CTX, in vitro, was able to reduce T cell proliferation, decreasing Th1 and Th17 and increasing Treg cell differentiation. Furthermore, in EAE model, the treatment with 5 consecutive doses of CTX inhibited IFN-γ-producing T cells, GM-CSF-producing T cells, reduced the frequency of activated microglia/macrophages within the CNS and decreased the number of migrating cell to spinal cord and cerebellum at the peak of the disease. These results suggest that CTX is a potential treatment not only for pain alteration but also for clinical progression induced by the disease as well as an useful tool for the development of new therapeutic approaches for the multiple sclerosis control.

Conserved Luminal C-Terminal Domain Dynamically Controls Interdomain Communication in Sarcolipin.

Sarcolipin (SLN) mediates Ca2+ transport and metabolism in muscle by regulating the activity of the Ca2+ pump SERCA. SLN has a conserved luminal C-terminal domain that contributes to its functional divergence among homologous SERCA regulators, but the precise mechanistic role of this domain remains poorly understood. We used all-atom molecular dynamics (MD) simulations of SLN totaling 77.5 µs to show that the N- (NT) and C-terminal (CT) domains function in concert. Analysis of the MD simulations showed that serial deletions of the SLN C-terminus do not affect the stability of the peptide nor induce dissociation of SLN from the membrane but promote a gradual decrease in both the tilt angle of the transmembrane helix and the local thickness of the lipid bilayer. Mutual information analysis showed that the NT and CT domains communicate with each other in SLN and that interdomain communication is partially or completely abolished upon deletion of the conserved segment Tyr29-Tyr31 as well as by serial deletions beyond this domain. Phosphorylation of SLN at residue Thr5 also induces changes in the communication between the CT and NT domains, which thus provides additional evidence for interdomain communication within SLN. We found that interdomain communication is independent of the force field used and lipid composition, which thus demonstrates that communication between the NT and CT domains is an intrinsic functional feature of SLN. We propose the novel hypothesis that the conserved C-terminus is an essential element required for dynamic control of SLN regulatory function.

Atomistic Structure and Dynamics of the Ca2+-ATPase Bound to Phosphorylated Phospholamban.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential components of the cardiac Ca2+ transport machinery. PLB phosphorylation at residue Ser16 (pSer16) enhances SERCA activity in the heart via an unknown structural mechanism. Here, we report a fully atomistic model of SERCA bound to phosphorylated PLB and study its structural dynamics on the microsecond time scale using all-atom molecular dynamics simulations in an explicit lipid bilayer and water environment. The unstructured N-terminal phosphorylation domain of PLB samples different orientations and covers a broad area of the cytosolic domain of SERCA but forms a stable complex mediated by pSer16 interactions with a binding site formed by SERCA residues Arg324/Lys328. PLB phosphorylation does not affect the interaction between the transmembrane regions of the two proteins; however, pSer16 stabilizes a disordered structure of the N-terminal phosphorylation domain that releases key inhibitory contacts between SERCA and PLB. We found that PLB phosphorylation is sufficient to guide the structural transitions of the cytosolic headpiece that are required to produce a competent structure of SERCA. We conclude that PLB phosphorylation serves as an allosteric molecular switch that releases inhibitory contacts and strings together the catalytic elements required for SERCA activation. This atomistic model represents a vivid atomic-resolution visualization of SERCA bound to phosphorylated PLB and provides previously inaccessible insights into the structural mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.