Basis for Accurate Protein pKa Prediction with Machine Learning.

pH regulates protein structures and the associated functions in many biological processes via protonation and deprotonation of ionizable side chains where the titration equilibria are determined by pKa's. To accelerate pH-dependent molecular mechanism research in the life sciences or industrial protein and drug designs, fast and accurate pKa prediction is crucial. Here we present a theoretical pKa data set PHMD549, which was successfully applied to four distinct machine learning methods, including DeepKa, which was proposed in our previous work. To reach a valid comparison, EXP67S was selected as the test set. Encouragingly, DeepKa was improved significantly and outperforms other state-of-the-art methods, except for the constant-pH molecular dynamics, which was utilized to create PHMD549. More importantly, DeepKa reproduced experimental pKa orders of acidic dyads in five enzyme catalytic sites. Apart from structural proteins, DeepKa was found applicable to intrinsically disordered peptides. Further, in combination with solvent exposures, it is revealed that DeepKa offers the most accurate prediction under the challenging circumstance that hydrogen bonding or salt bridge interaction is partly compensated by desolvation for a buried side chain. Finally, our benchmark data qualify PHMD549 and EXP67S as the basis for future developments of protein pKa prediction tools driven by artificial intelligence. In addition, DeepKa built on PHMD549 has been proven an efficient protein pKa predictor and thus can be applied immediately to, for example, pKa database construction, protein design, drug discovery, and so on.

Case report: subacute thyroiditis after receiving SARS-CoV-2 vaccine, maybe not only adjuvants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced the new coronavirus disease 2019 (COVID-19) pandemic worldwide. SARS-CoV-2 vaccines are designed to control the transmission of the disease. However, post-vaccination subacute thyroiditis (SAT) also appears with increase vaccination rate. Three cases of SAT after SARS-CoV-2 vaccines are described in this study. We have reported the patients' clinical symptoms, laboratory tests, and thyroid imaging. Tests for COVID-19 were all negative, and the patients did not report thyroid-related diseases, autoimmune diseases, or preceding upper respiratory system infections in their medical history. Three female patients showed neck pain on physical examination. The laboratory test results and imaging findings were consistent with the diagnostic criteria of SAT. The patients were carried out a standardized treatment according to their symptoms, and we closely followed up their response to the treatment. Clinicians must be aware of the possibility of SAT after receiving the vaccines and make timely therapy.

Selective Interactions of Mouse Melanocortin Receptor Accessory Proteins with Somatostatin Receptors.

Somatostatin receptors (SSTRs) are G protein-coupled receptors (GPCRs) known to regulate exocrine secretion, neurotransmission, and inhibit endogenous cell proliferation. SSTR subtypes (SSTR1-SSTR5) exhibit homo- or heterodimerization with unique signaling characteristics. Melanocortin receptor accessory protein 1 (MRAP1) functions as an allosteric modulator of melanocortin receptors and some other GPCRs. In this study, we investigated the differential interaction of MRAP1 and SSTRs and examined the pharmacological modulation of MRAP1 on mouse SSTR2/SSTR3 and SSTR2/SSTR5 heterodimerization in vitro. Our results show that the mouse SSTR2 forms heterodimers with SSTR3 and SSTR5 and that MRAP1 selectively interacts with SSTR3 and SSTR5 but not SSTR2. The interactive binding sites of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 locate on SSTR3 and SSTR5 but not SSTR2. The binding sites of MRAP1 to SSTR3 are extensive, while the ones of SSTR5 are restricted on transmembrane region six and seven. The heterodimerization of mouse SSTR2, SSTR3, and SSTR5 can be modulated by binding protein in addition to an agonist. Upregulation of extracellular signal-regulated kinases phosphorylation, p27Kip1, and increased cell growth inhibition with the co-expression of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 suggest a regulatory effect of MRAP1 on anti-proliferative response of two SSTR heterodimers. Taken together, these results provide a new insight of MRAP1 on the maintenance and regulation of mouse SSTR dimers which might be helpful to better understand the molecular mechanism involving SSTRs in tumor biology or other human disorders.

Pharmacological Evaluation of Melanocortin 2 Receptor Accessory Protein 2 on Axolotl Neural Melanocortin Signaling.

The Melanocortin-3 receptor (MC3R) and Melanocortin-4 receptor (MC4R), two members of the key hypothalamic neuropeptide signaling, function as complex mediators to control the central appetitive and energy homeostasis. The melanocortin 2 receptor accessory protein 2 (MRAP2) is well-known for its modulation on the trafficking and signaling of MC3R and MC4R in mammals. In this study, we cloned and elucidated the pharmacological profiles of MRAP2 on the regulation of central melanocortin signaling in a relatively primitive poikilotherm amphibian species, the Mexican axolotl (Ambystoma mexicanum). Our results showed the higher conservation of axolotl mc3r and mc4r across species than mrap2, especially the transmembrane regions in these proteins. Phylogenetic analysis indicated that the axolotl MC3R/MC4R clustered closer to their counterparts in the clawed frog, whereas MRAP2 fell in between the reptile and amphibian clade. We also identified a clear co-expression of mc3r, mc4r, and mrap2 along with pomc and agrp in the axolotl brain tissue. In the presence of MRAP2, the pharmacological stimulation of MC3R by α-MSH or ACTH significantly decreased. MRAP2 significantly decreased the cell surface expression of MC4R in a dose dependent manner. The co-localization and formation of the functional complex of axolotl MC3R/MC4R and MRAP2 on the plasma membrane were further confirmed in vitro. Dramatic changes of the expression levels of mc3r, mrap2, pomc, and agrp in the fasting axolotl hypothalamus indicated their critical roles in the metabolic regulation of feeding behavior and energy homeostasis in the poikilotherm aquatic amphibian.

Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by the C-Terminus of MRAP2 Protein.

Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts on its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate appetite and energy homeostasis. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine modulator of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal activities and peripheral energy balance. Here, we demonstrated the interaction between MRAP2 and MCHR1 by immunoprecipitation and bimolecular fluorescent assay and found that MRAP2 could inhibit MCHR1 signaling in vitro. A series of functional truncations of different regions further identified that the C-terminal domains of MRAP2 protein were required for the pharmacological modulation of intracellular Ca2+ coupled cascades and membrane transport. These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

Identification of MRAP protein family as broad-spectrum GPCR modulators.

BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.

Functional Characterization of the Internal Symmetry of MRAP2 Antiparallel Homodimer.

The melanocortin receptors are defined as a series of vital pharmaceutical targets to regulate neuronal appetite and maintain controllable body weight for mammals and teleosts. Melanocortin receptor accessory protein 2 (MRAP2) functions as an essential accessory player that modulates the surface translocation and binding to a variety of endogenous or synthetic hormones of central melanocortin-4 receptor (MC4R) signaling. MRAP2 is a single-transmembrane protein and could form a functional symmetric antiparallel homodimer topology. Here, we inverted the N-terminal, transmembrane, and C-terminal domains and generated six distinct conformational variants of the mouse MRAP2 to explore the functional orientations and the internal symmetry of MRAP2 dimers. These remolded MRAP2 mutants showed proper assembly of the antiparallel homodimer and binding to the MC4R, but slightly altered the regulatory profile on the surface expression and the ligand-stimulated cAMP cascades of MC4R. This study elucidated the importance of the orientation of each domain of the single-transmembrane protein and revealed the pharmacological properties of the internal symmetry of the antiparallel homodimer for MRAP2.

Identification of novel GPCR partners of the central melanocortin signaling.

OBJECTIVE: Homo- or heterodimerization of G protein-coupled receptors (GPCRs) generally alters the normal functioning of these receptors and mediates their responses to a variety of physiological stimuli in vivo. It is well known that melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) are key regulators of appetite and energy homeostasis in the central nervous system (CNS). However, the GPCR partners of MC3R and MC4R are not well understood. Our objective is to analyze single cell RNA-seq datasets of the hypothalamus to explore and identify novel GPCR partners of MC3R and MC4R and examine the pharmacological effect on the downstream signal transduction and membrane translocation of melanocortin receptors. METHODS: We conducted an integrative analysis of multiple single cell RNA-seq datasets to reveal the expression pattern and correlation of GPCR families in the mouse hypothalamus. The emerging GPCRs with important metabolic functions were selected for cloning and co-immunoprecipitation validation. The positive GPCR partners were then tested for the pharmacological activation, competitive binding assay and surface translocation ELISA experiments. RESULTS: Based on the expression pattern of GPCRs and their function enrichment results, we narrowed down the range of potential GPCR interaction with MC3R and MC4R for further confirmation. Co-immunoprecipitation assay verified 23 and 32 novel GPCR partners that interacted with MC3R and MC4R in vitro. The presence of these GPCR partners exhibited different effects in the physiological regulation and signal transduction of MC3R and MC4R. CONCLUSIONS: This work represented the first large-scale screen for the functional GPCR complex of central melanocortin receptors and defined a composite metabolic regulatory GPCR network of the hypothalamic nucleuses.

Studies on Pitting Corrosion of Al-Cu-Li Alloys Part II: Breakdown Potential and Pit Initiation.

Prediction of the accumulated pitting corrosion damage in aluminum-lithium (Al-Li) is of great importance due to the wide application of these alloys in the aerospace industry. The Point Defect Model (PDM) is arguably one of the most well-developed techniques for evaluating the electrochemical behavior of passive metals. In this paper, the passivity breakdown and pitting corrosion performance of AA 2098-T851 was investigated using the PDM with the potentiodynamic polarization (PDP) technique in NaCl solutions at different scan rates, Cl- concentrations and pH. Both the PDM predictions and experiments reveal linear relationships between the critical breakdown potential (Ec) of the alloy and various independent variables, such as aCl- and pH. Optimization of the PDM of the near-normally distributed Ec as measured in at least 20 replicate experiments under each set of conditions, allowing for the estimation of some of the critical parameters on barrier layer generation and dissolution, such as the critical areal concentration of condensed cation vacancies (ξ) at the metal/barrier layer interface and the mean diffusivity of the cation vacancy in the barrier layer (D). With these values obtained-using PDM optimization-in one set of conditions, the Ec distribution can be predicted for any other set of conditions (combinations of aCl-, pH and T). The PDM predictions and experimental observations in this work are in close agreement.

Studies on Pitting Corrosion of Al-Cu-Li Alloys Part III: Passivation Kinetics of AA2098-T851 Based on the Point Defect Model.

In this paper, the passivation kinetics of AA2098-T851 was investigated by a fundamental theoretical interpretation of experimental results based on the mixed potential model (MPM). The steady state passive layer formed on the AA2098-T851 in NaHCO3 solution in a CO2 atmosphere upon potentiostatic stepping in the anodic direction followed by stepping in the opposite direction was explored. Potentials were selected in a way that both anodic passive dissolution of the metal and hydrogen evolution reaction (HER) occur, thereby requiring the MPM for interpretation. Optimization of the MPM on the experimental electrochemical impedance spectroscopy (EIS) data measured after each potentiostatic step revealed the important role of the migration of Al interstitials in determining the kinetics of passive layer formation and dissolution. More importantly, it is shown that the inequalities of the kinetics of formation and dissolution of the passive layer as observed in opposite potential stepping directions lead to the irreversibility of the passivation process. Finally, by considering the Butler-Volmer (B-V) equation for the cathodic reaction (HER) in the MPM, and assuming the quantum mechanical tunneling of the charge carriers across the barrier layer of the passive film, it was shown that the HER was primarily controlled by the slow electrochemical discharge of protons at the barrier layer/solution (outer layer) interface.

Temperature and initial composition dependence of pattern formation and dynamic behavior in phase separation under deep-quenched conditions.

Phase separation of SCN-H2O ([CH2CN]2-H2O) transparent solutions is simulated in two dimensions and the effects of quenching temperature and initial composition on the pattern formation and dynamic behavior of the second phase are examined via Minkowski functionals. The simulation is based on model H where the molar free energy of the SCN-H2O solution is obtained by the CALPHAD approach. We find that the composition and temperature do not affect the exponent in the domain growth law, where the average domain size with time yields R(t)-t n . However, they influence the pattern formation and dynamic behavior of the second phase in phase separation. Lower temperature leads to a finer bicontinuous structure in spinodal decomposition and promotes the nucleation rate, which accelerates the phase separation and results in more liquid droplets with smaller size. As the initial composition diverges from the critical value, the spatial patterns change gradually from bicontinuous into a droplet-like structure. When the initial composition is closer to the critical value, for spinodal decomposition, the diffusion-driven growth lasts for a longer time and the average domain size of liquid droplets is larger. For nucleation-driven growth, in contrast, the single phase separates more quickly and the average size of liquid droplets is smaller.

Studies on Pitting Corrosion of Al-Cu-Li Alloys Part I: Effect of Li Addition by Microstructural, Electrochemical, In-situ, and Pit Depth Analysis.

To analyze the effect of lithium and microstructure on the pitting corrosion behavior of aluminum alloys, three types of aluminum alloys were studied via scanning electron microscopy, transmission electron microscopy, electrochemical polarization, and by immersion tests coupled with in-situ observation of pitting and statistical analysis of pit depths measured by surface profilometry. It was found that, with increasing lithium content, the resistance to pitting corrosion was enhanced and the passive range was enlarged. In-situ observation revealed that the development of pitting corrosion exhibited three stages, including an initial slow nucleation stage (Stage I), a fast development stage (Stage II), and a stabilized growth stage (Stage III). Higher lithium content contributed to shorter time periods of Stages I and II, resulting in faster pitting evolution and a higher number of pits. However, the pits were generally shallower for the specimen with the highest lithium content, which is in agreement with the results of the electrochemical analysis.