Cooperative binding mitigates the high-dose hook effect

Background: The high-dose hook effect (also called prozone effect) refers to the observation that if a multivalent protein acts as a linker between two parts of a protein complex, then increasing the amount of linker protein in the mixture does not always increase the amount of fully formed complex. On the contrary, at a high enough concentration range the amount of fully formed complex actually decreases. It has been observed that allosterically regulated proteins seem less susceptible to this effect. The aim of this study was two-fold: First, to investigate the mathematical basis of how allostery mitigates the prozone effect. And second, to explore the consequences of allostery and the high-dose hook effect using the example of calmodulin, a calcium-sensing protein that regulates the switch between long-term potentiation and long-term depression in neurons. Results: We use a combinatorial model of a “perfect linker protein” (with infinite binding affinity) to mathematically describe the hook effect and its behaviour under allosteric conditions. We show that allosteric regulation does indeed mitigate the high-dose hook effect. We then turn to calmodulin as a real-life example of an allosteric protein. Using kinetic simulations, we show that calmodulin is indeed subject to a hook effect. We also show that this effect is stronger in the presence of the allosteric activator Ca 2+/calmodulin-dependent kinase II (CaMKII), because it reduces the overall cooperativity of the calcium-calmodulin system. It follows that, surprisingly, there are conditions where increased amounts of allosteric activator actually decrease the activity of a protein. Conclusions: We show that cooperative binding can indeed act as a protective mechanism against the hook effect. This will have implications in vivo where the extent of cooperativity of a protein can be modulated, for instance, by allosteric activators or inhibitors. This can result in counterintuitive effects of decreased activity with increased concentrations of both the allosteric protein itself and its allosteric activators. Electronic supplementary material The online version of this article (doi:10.1186/s12918-017-0447-8) contains supplementary material, which is available to authorized users

Allosteric regulation of synaptic processes

Glutamatergic neurotransmission is of key importance for short-term and long-term plasticity in the hippocampus, a part of the medial temporal lobe which is responsible for processes of explicit semantic and spatial memory. Short-term plasticity is mainly regulated by the presynaptic neuron and long-term plasticity is to large parts regulated by the post-synaptic neuron. In this thesis we have looked into cellular and molecular biophysical mechanisms in glutamatergic neurons mainly in the hippocampus. We first reviewed the presynaptic mechanisms underlying short-term plasticity like assembly of the release machinery, positional and molecular priming, site preparation, calcium dynamics regulation, intrinsic vesicular fusogenicity, endocytosis, acidification and filling. In study 1 we looked into the role of intrinsic vesicle fusogenicity on short-term plasticity by formulating a deterministic vesicular release model based on ordinary differential equations. Intrinsic vesicular fusogenicity was an allosteric property we invented in order to test the hypothesis of calcium independence. The model was able to simulate properties of resting neurons, by reproducing the spontaneous release rates and the size of the readily releasable pool. Furthermore, assuming that the heterogeneity in vesicular release probability arises due to differences in intrinsic vesicular fusogenicity, the model was able to explain depression by an imbalance between fusion and vesicular priming. It also predicted that facilitation could be due to an increase in intrinsic vesicular fusogenicity, which together with build-up of calcium gave rise to initial increase in vesicular release. Finally, we investigated the effect of three different modes of regulation of release probability on short-term plasticity. It was seen that differences in intrinsic vesicular fusogenicity gave rise to a more significant change in shortterm plasticity than change in calcium sensitivity of release. All in all the results tell us that intrinsic vesicular fusogenicity has an important role in tuning short-term plasticity. In study 2 we investigated the regulation of the postsynaptic allosteric AMPA receptor. To do this we developed a model based on the Monod Wyman Changeux framework which described the ligand concentration dependence of the conductance states by increasing affinity to conductance states. The model was able to explain thermodynamic behaviours of native and recombinant receptors when stimulated with full agonists like glutamate and quisqualate as well as partial agonists like willardiines. It was also predicted that the receptor stabilizes its large conductance state within the rise time of a so-called 'mini' post-synaptic current, providing a possible underlying mechanism for the peak of the current. In study 3 we investigated the high-dose hook effect in allosteric proteins by first developing a combinatorical theory for how linker proteins behave under conditions of perfect binding. The theory predicted that the steady-state concentration of fully bound linker-proteins decreases at a critical concentration of initial free linker protein as the free linker protein concentration is increased. This effect is however decreased in proteins where binding of ligand occurs in a cooperative fashion. The outcome was validated by simulations of dimeric and tetrameric linker proteins under imperfect binding. We also simulated the cooperative synaptic protein calmodulin, and it was seen to be subject to the hook effect. The hook effect was stronger in the presence of the allosteric activator Ca2+/calmodulin kinase II (CamKII). We show that increased amounts of the allosteric activator can decrease the activity of calmodulin. At 140 uM calmodulin behaved only as if the molecule only appeared in the relaxed (R) state. The relaxed state has no cooperativity, but has higher ligand affinity than the wild-type calmodulin. Even though this phenomenon may be present in many different biochemical systems, synapses contain several linker proteins that are pivotal for synaptic plasticity for instance AMPA receptors, synaptotagmin, calbinding and calmodulin. In summary, this thesis gives insight into allosteric mechanisms in glutamatergic hippocampal neurons by using whole-cell voltage clamp and algebraic modelling. Specifically, it suggests an explanation for the important role of allosteric mechanisms in vesicular release probability and short-term plasticity. It also provides an explanation for the ligand concentration dependence of AMPA receptors and puts forward a theory for how complexes and active forms of linker proteins behave under increase of free linker protein concentration, a behaviour might contribute to pre-and postsynaptic processes

Celastrol: A Spectrum of Treatment Opportunities in Chronic Diseases

This deposit is composed by the main article, and it hasn't any supplementary materials associated.The identification of new bioactive compounds derived from medicinal plants with significant therapeutic properties has attracted considerable interest in recent years. Such is the case of the Tripterygium wilfordii (TW), an herb used in Chinese medicine. Clinical trials performed so far using its root extracts have shown impressive therapeutic properties but also revealed substantial gastrointestinal side effects. The most promising bioactive compound obtained from TW is celastrol. During the last decade, an increasing number of studies were published highlighting the medicinal usefulness of celastrol in diverse clinical areas. Here we systematically review the mechanism of action and the therapeutic properties of celastrol in inflammatory diseases, namely, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, osteoarthritis and allergy, as well as in cancer, neurodegenerative disorders and other diseases, such as diabetes, obesity, atherosclerosis, and hearing loss. We will also focus in the toxicological profile and limitations of celastrol formulation, namely, solubility, bioavailability, and dosage issues that still limit its further clinical application and usefulness.Fundação para a Ciência e a Tecnologia grants: (SFRH/BPD/92860/2013, PTDC/BIM-MEC/4665/2014); European Research Council grants: (ERC-2014-CoG 647888-iPROTECTION).info:eu-repo/semantics/publishedVersio

AZITHROMYCIN THERAPY REDUCES CARDIAC INFLAMMATION AND MITIGATES ADVERSE CARDIAC REMODELING AFTER MYOCARDIAL INFARCTION

Introduction: Myocardial infarction (MI) remains the leading cause of morbidity and mortality worldwide. Induced by cardiomyocyte death, MI initiates a prolonged and uncontrolled inflammatory response which impairs the healing process. Immune cells, such as macrophages, play a central role in organizing the early post-MI inflammatory response and the subsequent repair phase. Two activation states of macrophages have been identified with distinct and complementary functions (inflammatory vs. reparatory). This bimodal pattern of macrophage activation is an attractive therapeutic target to favorably resolve post-MI inflammation and enhance recovery. It has been demonstrated that azithromycin (AZM), a commonly used antibiotic with immunomodulatory effects, polarizes macrophages towards the reparatory phenotype. AZM has an excellent safety profile and has been approved for human use. We hypothesize that AZM reduces inflammation and improves heart function in MI. Methods and results: In our initial studies, we demonstrated that oral free AZM (160 mg/kg daily for 7 days), initiated 3 days prior to MI, enhances post-MI cardiac recovery as a result of shifting macrophages to the reparatory state. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory and an increase in reparative macrophages, decreasing the pro-inflammatory/reparative macrophage ratio. Macrophage changes were associated with a significant decline in pro- and an increase in anti-inflammatory cytokines. Additionally, AZM treatment was correlated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the reparative phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis. We designed this proof of concept study using pre-MI AZM therapy to achieve steady state levels prior to injury. Therefore, in our follow-up studies we targeted inflammatory macrophages using a non-Pegylated liposomal formulation of AZM (Lazm) which has been shown in multiple studies to promote drug efficacy and minimize off-target effects. To test the hypothesis that Lazm is more effective and safer than free AZM, low doses of free/liposomal AZM (10 or 40 mg/kg, administered intravenously) were initiated immediately after MI. We observed that Lazm induces early resolution of the post-MI inflammatory response as evidenced by switching of the activation state of monocytes/macrophages towards the reparatory phenotype. Neutrophils were substantially decreased, particularly pro-inflammatory neutrophils. Cytokine profiles were also shifted to the anti-inflammatory status with Lazm therapy. Taken together, AZM treatment resulted in a significant shift in macrophage activation towards the reparatory state. The shift in inflammatory state was accompanied by a decrease in apoptosis and infarct size in the injured heart, as well as enhanced angiogenesis and LV functional recovery in our long-term studies. In addition, Lazm was protective against off-target effects of AZM on the heart. Conclusion: This is the first evidence of a novel and clinically-relevant therapeutic strategy to modulate post-MI inflammation. We found that AZM reduces cardiac inflammation and improves adverse cardiac remodeling after infarction via promoting a shift of macrophage activation state. The overarching significance of this work is the modulation of sterile inflammation, which can be a viable therapeutic target in many conditions including stroke and heart attack. Additionally, this is the first study to demonstrate the immune modulation properties of liposomal AZM, which has wide potential therapeutic applications beyond the cardiovascular field. Importantly, liposomal formulation of AZM is protective from its cardiac off-target effects. Our findings strongly support clinical trials using AZM as a novel and clinically relevant therapeutic target to improve cardiac recovery and reduce heart failure post-MI in humans

Celastrol: A Spectrum of Treatment Opportunities in Chronic Diseases

The identification of new bioactive compounds derived from medicinal plants with significant therapeutic properties has attracted considerable interest in recent years. Such is the case of the Tripterygium wilfordii (TW), an herb used in Chinese medicine. Clinical trials performed so far using its root extracts have shown impressive therapeutic properties but also revealed substantial gastrointestinal side effects. The most promising bioactive compound obtained from TW is celastrol. During the last decade, an increasing number of studies were published highlighting the medicinal usefulness of celastrol in diverse clinical areas. Here we systematically review the mechanism of action and the therapeutic properties of celastrol in inflammatory diseases, namely, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, osteoarthritis and allergy, as well as in cancer, neurodegenerative disorders and other diseases, such as diabetes, obesity, atherosclerosis, and hearing loss. We will also focus in the toxicological profile and limitations of celastrol formulation, namely, solubility, bioavailability, and dosage issues that still limit its further clinical application and usefulness

PROTACs – A Novel and Rapidly Developing Field of Targeted Protein Degradation

There is a continued need for new technology and strategies for tackling cancer and other diseases, and within the current century a novel therapeutic strategy has emerged in the realm of targeted protein degradation called Proteolysis-Targeting Chimeras (PROTACs). This technology specifically targets and degrades disease-causing proteins via the ubiquitin-proteasome system, and has seen an explosion of research and intrigue in both academia and industry over the past two decades. The diversity of PROTAC classes based on the E3 ligase recruiting ligand and the target protein allows for a universal molecular structure that can be customized for a specific target and disease. While it is primarily heavily focused in the realm of cancer therapeutics, PROTACs have expanded into other diseases such as cardiovascular, neurodegenerative, and virus-caused diseases. The discovery of novel PROTAC designs also allows for the field to overcome its own shortcomings and develop into new directions. Overall, the intrigue of PROTAC technology’s ability to degrade ‘undruggable’ targets has driven the field of research to expand rapidly in the short time since its initial discovery and continued intense efforts will help further shape the field to transition into the clinical setting to benefit the world

The role of sarcoplasmic calcium in skeletal muscle training adaptation

Current research shows a clear correlation between strong mitochondrial capacity, healthy muscle and general public health. A sedentary lifestyle increases the risk of a whole host of so called ‘western diseases’, while an active lifestyle reduce the risk of said diseases. Thus, well-functioning muscles are a necessity for general health. So far endurance exercise is the most effective method to improve muscle function. This thesis will focus on the cellular mechanisms that regulate muscle performance and how these can be improved. In the first study, we show that supplemented dietary nitrate enhances Ca2+ handling and submaximal force in mouse fast twitch muscle. Continuing this, in study two, we show that the increased submaximal force enhances voluntary activity in mice, presumably due to a shifted perceived effort of running. In study three we show that mild stress from cold exposure can enhance mitochondrial biogenesis resulting in improved fatigue resistance without exercise. The cold environment seems to induce a sarcoplasmic reticulum (SR) Ca2+ leak in the skeletal muscle. In study four we investigated why short (180s) high intensity interval training works better for enhancing endurance than regular low-intensity exercise. We show that oxidants formed during exercise causes ryanodine receptor modifications, which result in a SR Ca2+ leak and this in turn likely triggers transcription to improved mitochondrial capacity. In study five we show that inducing a mild SR Ca2+ leak, either with exercise or pharmacological tools, drive mitochondrial biogenesis. In study six we show that a in a model of ageing, a degenerative mitochondrial problem causes myopathy via reduced SR Ca2+ release. Ca2+ is a central player in muscle function. This thesis shows that diet, exercise and age have the ability to affect skeletal muscle Ca2+ handling. Most importantly, Ca2+ signals can improve mitochondrial function, resulting in improved muscle function. However degenerative mitochondria causes reduced Ca2+handling that leads to muscle weakness. This is one of the reasons an active lifestyle is so important for the elderly, because it improves the mitochondrial function rather than being degraded. Perhaps in the future, inducing a small SR Ca2+ leak could minimize some of the risks associated with sedentary lifestyle

Streptococcal collagen-like protein 1, Scl1, modulates group a Streptococcus adhesion, biofilm formation and virulence

Background: The collagens comprise a large family of versatile proteins found in all three domains of life. The streptococcal collagen-like protein 1, scl1, of group A Streptococcus (GAS) binds extracellular matrix components (ECM), cellular fibronectin and laminin, via the surface-exposed globular domain. GAS strains express scl1 and form biofilm in vitro, except for M3-type strains that are particularly invasive to humans. Hypothesis: Lack of scl1 adhesin in M3 GAS results in decreased adherence and biofilm formation, and increased virulence. Results and Discussion : First crystal structure of the globular domain revealed a unique six-helical bundle fold, consisting of three pairs of alpha helices connected by variable loops. ECM binding by Scl1 promotes the formation of stable tissue microcolonies, which was demonstrated in vitro during infection of wounded human skin equivalents. A conserved nonsense mutation was identified in the scl1 allele of the M3-type strains (scl1.3) that truncates the coding sequence, presumably resulting in a secreted Scl1 variant. Absence of Scl1 on the surface of M3-type GAS was demonstrated experimentally, as well as diminished expression of the scl1 transcript in M3 strains relative to other M-types. Therefore, M3-type strains have reduced biofilm capacity on ECM coatings relative to other M-types. Constructed full-length recombinant Scl1.3 protein displayed binding capacity to cellular fibronectin and laminin, and M3 strains complemented with functional Scl1.3 adhesin displayed increased biofilm formation. The isoallelic M3 strain, carrying a rare carrier allele encoding cell-associated Scl1.3 variant, showed decreased pathology in mice, compared to the invasive M3 strain. Similarly, scl1 inactivation in biofilm-capable M28- and M41-type GAS led to increased lesion size during subcutaneous infection. Conclusions: The studies presented here demonstrate the importance of surface Scl1 in modulating biofilm formation and virulence of GAS, and provide insight into the structure and function of Scl proteins

Designing proteasome adaptors to deplete specific proteins from cells

Cellular protein levels are governed by their rates of synthesis and degradation, and both processes are intricately regulated. One way to study the role of a protein in the cell is to artificially deplete it and observe the effects. The most common methodology for depleting proteins inhibits expression of the target through RNA interference. However, this technique acts at the protein synthesis level and cannot be used to study long-lived proteins or post-translational modifications. Thus, a complementary approach that acts on the proteins themselves would be useful. One method is to target a protein to the cell’s degradation machinery, the Ubiquitin Proteasome System (UPS). Proteins are targeted to the proteasome by the covalent attachment of ubiquitin molecules, which are recognized by the proteasome. The substrate is then translocated into the proteasome’s proteolytic core and degraded into short peptides, while the ubiquitin molecules are cleaved off and recycled. Recently, methods have been developed to deplete proteins by inducing their ubiquitination, which accelerates their degradation by the proteasome. Ubiquitin is a signaling molecule for numerous cellular pathways other than proteolysis, however, and inducing ubiquitination does not always lead to degradation. Therefore, I have developed a system to degrade specific proteins in cells using chimeric adaptors that shuttle proteins directly to the proteasome without the need for ubiquitination. I have shown that this system can be successfully applied to several proteins in vitro and that the adaptors can induce degradation of model and endogenous proteins in cells.Cellular and Molecular Biolog