p38 MAPK signaling in chronic obstructive pulmonary disease pathogenesis and inhibitor therapeutics.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by persistent respiratory symptoms and airflow limitation due to airway and/or alveolar remodeling. Although the abnormalities are primarily prompted by chronic exposure to inhaled irritants, maladjusted and self-reinforcing immune responses are significant contributors to the development and progression of the disease. The p38 isoforms are regarded as pivotal hub proteins that regulate immune and inflammatory responses in both healthy and disease states. As a result, their inhibition has been the subject of numerous recent studies exploring their therapeutic potential in COPD. MAIN BODY: We performed a systematic search based on the PRISMA guidelines to find relevant studies about P38 signaling in COPD patients. We searched the PubMed and Google Scholar databases and used "P38" AND "COPD" Mesh Terms. We applied the following inclusion criteria: (1) human, animal, ex vivo and in vitro studies; (2) original research articles; (3) published in English; and (4) focused on P38 signaling in COPD pathogenesis, progression, or treatment. We screened the titles and abstracts of the retrieved studies and assessed the full texts of the eligible studies for quality and relevance. We extracted the following data from each study: authors, year, country, sample size, study design, cell type, intervention, outcome, and main findings. We classified the studies according to the role of different cells and treatments in P38 signaling in COPD. CONCLUSION: While targeting p38 MAPK has demonstrated some therapeutic potential in COPD, its efficacy is limited. Nevertheless, combining p38 MAPK inhibitors with other anti-inflammatory steroids appears to be a promising treatment choice. Clinical trials testing various p38 MAPK inhibitors have produced mixed results, with some showing improvement in lung function and reduction in exacerbations in COPD patients. Despite these mixed results, research on p38 MAPK inhibitors is still a major area of study to develop new and more effective therapies for COPD. As our understanding of COPD evolves, we may gain a better understanding of how to utilize p38 MAPK inhibitors to treat this disease. Video Abstract.

We wanted to determine what studies have been done on how a protein called p38 affects a lung disease called COPD. COPD is a condition that makes it hard to breathe and can cause coughing, wheezing, and chest infections. p38 is a protein that helps cells to respond to stress and inflammation, but it may also play a role in causing or worsening COPD. We searched two main online databases for studies that met our criteria. We looked for studies that involved humans, studies that used animals or cells in the lab, studies that reported new findings, studies that were written in English, and studies that focused on p38 and COPD. We did not include studies that were reviews, summaries, opinions, or letters or studies that were not related to p38 or COPD. We found 361 studies that matched our criteria. We read the titles and summaries of these studies and checked the full texts for quality and relevance. We collected information from each study, such as who did it, when and where it was done, how many people were involved, what type of cells were studied, what treatment was given, what outcome was measured, and what the main results were. We grouped the studies based on the type of cells and type of treatment they studied. We found that different types of cells (such as lung cells, immune cells, and blood cells) and different types of treatment can affect how p38 works in COPD.

Ion behavior in the selectivity filter of HCN1 channels.

Hyperpolarization-activated cyclic-nucleotide gated channels (HCNs) are responsible for the generation of pacemaker currents (If or Ih) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) channels, HCNs show much lower selectivity for K+ over Na+ ions. This increased permeability to Na+ is critical to their role in membrane depolarization. HCNs can also select between Na+ and Li+ ions. Here, we investigate the unique ion selectivity properties of HCNs using molecular-dynamics simulations. Our simulations suggest that the HCN1 pore is flexible and dilated compared with Kv channels with only one stable ion binding site within the selectivity filter. We also observe that ion coordination and hydration differ within the HCN1 selectivity filter compared with those in Kv and cyclic-nucleotide gated channels. Additionally, the C358T mutation further stabilizes the symmetry of the binding site and provides a more fit space for ion coordination, particularly for Li+.

Structure and dynamics of inactive and active MARK4: conformational switching through the activation process.

Microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine protein kinase belonging to a highly-conserved group of PAR proteins that phosphorylate microtubule-associated proteins (tau, MAP2 and MAP4) and regulate cell polarity. MARK4 isoform is mainly found in brain tissue, causing microtubule destabilization in neuronal cells and also tau-protein phosphorylation seen in Alzheimer's disease. In this study, the dynamic behavior of inactive and active structures of human MARK4 was studied by modeling and molecular dynamics simulations and motions of the protein through activation process were interpolated. Structure and dynamics of the protein active state in presence of Mg-ATP were also studied. The results suggest for occlusion of ATP binding site by activation loop, as the main inactivation mechanism. Data also justify the necessity of UBA (ubiquitin-associated) domain auto-inhibitory role. Within the inactive and active state, G-loop is highly fluctuating and assumes an open conformation and away from Mg-ATP complex. This behavior raises the assumption that this loop may experience other stabilizing interactions with residues out of the kinase core which help its stabilization during the phospho-transfer reaction. Mg-ATP complex localization is well preserved within the catalytic cleft through the electrostatic interactions. However, minor rearrangements of water molecules around this complex are allowed, which may further refine the delicate position of this complex for phospho-transfer reaction.Communicated by Ramaswamy H. Sarma.

MARK4 protein can explore the active-like conformations in its non-phosphorylated state.

Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules. In the current study, the non-phosphorylated conformation of the activation loop was modeled in a structure representing the enzymatically inactive form of this protein, and its dynamics were evaluated through a 100 ns initial all-atom simulation, which was prolonged by another 2 µs. Although the activation loop was folding on itself and was leaning toward ATP site in the initial modeled structure, soon after the initiating the simulation, this loop stretched away from the ATP binding site and stably settled in its new position for the rest of simulation time. A network of hydrogen bonds, mainly between the activation segment residues, αC-helix and the catalytic loop reinforced this conformation. Interestingly, several features of active kinase conformation such as formation of R-spine, Glu106-Lys88 salt-bridge, and DFG-In motif were observed during a considerable number of trajectory frames. However, they were not sustainably established during the simulation time, except for the DFG-In motif. Consequently, this study introduces a stable conformation of the non-phosphorylated form of MARK4 protein with a partially stretched activation loop conformation as well as partial formation of R-spine, closely resembling the active kinase.

Maltase-glucoamylase inhibition potency and cytotoxicity of pyrimidine-fused compounds: An in silico and in vitro approach.

The prevalence of diabetes mellitus has been incremented in the current century and the need for novel therapeutic compounds to treat this disease has been significantly increased. One of the most promising approaches is to inhibit intestinal alpha glucosidases. Based on our previous studies, four pyrimidine-fused heterocycles (PFH) were selected as they revealed satisfactory inhibitory action against mammalian α-glucosidase. The interaction of these compounds with both active domains of human maltase-glucoamylase (MGAM) and their effect on human Caco-2 cell line were investigated. The docking assessments suggested that binding properties of these ligands were almost similar to that of acarbose by establishing hydrogen bonds especially with Tyr1251 and Arg526 in both C-terminal and N-terminal MGAM, respectively. Also, these compounds indicated a stronger affinity for C-terminal of MGAM. L2 and L4 made tightly complexes with both terminals of MGAM which in turn revealed the importance of introducing pyrimidine scaffold and its hinge compartment. The results of molecular dynamics simulation analyses confirmed the docking data and showed deep penetration of L2 and L4 into the active site of MGAM. Based on cell cytotoxicity assessments, no significant cell death induction was observed. Hence, these functional MGAM inhibitors might be considered as new potential therapeutic compounds in treatment of diabetes and its complications.

Disulfide bridge formation to increase thermostability of DFPase enzyme: A computational study.

Organophosphate compounds bioremediation by use of organophosphorus degradation enzymes such as DFPase is a developing interest in industry and medicine. The most important problem with the bio-catalytic enzymes is their instability on high temperatures. This work carried out to find suitable locations for introducing disulfide bridges in DFPase enzyme. We employed some computational approaches to design the disulfide bridges and evaluate their roles in the enzyme structural thermostability. According to the in silico results, mutant 6 (V24C, C76) increased the enzyme thermostability relative to wild-type.

Application of molecular dynamics simulations to design a dual-purpose oligopeptide linker sequence for fusion proteins.

Proteins are often monitored by combining a fluorescent polypeptide tag with the target protein. However, due to the high molecular weight and immunogenicity of such tags, they are not suitable choices for combining with fusion proteins such as immunotoxins. In this study, we designed a polypeptide sequence with a dual role (it acts as both a linker and a fluorescent probe) to use with fusion proteins. Two common fluorescent tag sequences based on tetracysteine were compared to a commonly used rigid linker as well as our proposed dual-purpose sequence. Computational investigations showed that the dual-purpose sequence was structurally stable and may be a good choice to use as both a linker and a fluorescence marker between two moieties in a fusion protein.

Interconversion of inactive to active conformation of MARK2: Insights from molecular modeling and molecular dynamics simulation.

The Ser/Thr protein kinase MARK2, also known as Par1b, belongs to the highly-conserved family of PAR proteins which regulate cell polarity and partitioning through the animal kingdom. In the current study, inactive and active structures of human MARK2 were constructed by modeling and molecular dynamics simulation, based on available incomplete crystal structures in Protein Data Bank, to investigate local structural changes through which MARK2 switches from inactive to active state. None of the MARK2 wild type inactive crystal structures represent the position of activation segment. So, the contribution of this loop to the formation of inactive state is not clear. In the modeled structure of inactive MARK2, activation segment occludes the enzyme active site and assumes a relatively stable position. We also presented a detailed description of the major structural changes occur through the activation process and proposed a framework on how these deviations might be affected by the phosphorylation of Thr208 or existence of the UBA domain. Inspection of protein active state in the presence of Mg-ATP, demonstrated the precise arrangement of the various parts of enzyme around Mg-ATP and the importance of their stability in localization of the resulting complex. The results also confirmed the alleged mild auto-inhibitory role of the UBA domain and suggested a reason for the necessity of this role, based on structural similarities to other related kinases.

Rational design and engineering of a mutant variant of urate oxidase as a therapeutic enzyme: a molecular dynamics simulation approach.

BACKGROUND: Urate oxidase is absent in humans so the enzyme is considered as an important therapeutic agent to control hyperuricemic disorders. Currently available enzymes with pharmaceutical applications have adverse effects associated with allergic reactions and anaphylactic shocks, in case of chronic treatment. Therefore, developing variant forms of the enzyme, with lower immunogenicity and similar or higher activity, is of great importance. AIM: Here, we tried to improve the structure of a recently resurrected ancestral mammalian urate oxidase (which is claimed to have higher enzymatic activity compared to other mammalian counterparts) by introducing eight rational mutations and verified the consequence of these mutations on immunogenicity, stability and the affinity of protein to uric acid by computational techniques. METHODS: After modeling the full-length wild-type and mutant structures, structural dynamics were monitored through 20 ns and 50 ns of molecular dynamics simulation by GROMACS software package for the structures holding and lacking uric acid, respectively. RESULTS: Simulation results implied maintenance of 3D arrangement, volume and compactness between wild-type and mutant structures. However residues of the mutated structure showed a higher tendency for hydrogen bond formation leading to a more stable and more soluble protein package with a higher surface area buried between protein chains. We also used the DiscoTope-2.0 server to map changes in immunogenicity index of 50 structures derived from the last 10ns of simulation. CONCLUSION: Finally, this study suggests a urate oxidase mutant with improved overall stability, reduced immunogenicity and slightly lower affinity for uric acid compared to the resurrected ancestral mammalian urate oxidase.

Comparison of zn, cu, and fe content in hair and serum in alopecia areata patients with normal group.

Background. Alopecia areata (AA) is an autoimmune condition, in which hair is lost from some areas of the body. Though its etiopathogenesis is not fully understood, there are claims that imbalance of trace elements may trigger the onset of AA, by distorting immune functions. In this study, we tried to investigate the relationship between AA and iron, zinc, and copper levels of serum and hair. Materials and Methods. Sixteen female patients with AA (14-40 years old) and 27 healthy female controls were enrolled in this study. Serum and hair level of iron, zinc, and copper were measured by flame emission spectroscopy. The resulting data was analyzed with SPSS15. Results. We did not detect a significant difference in the serum and hair level of iron, zinc, and copper between patients and controls. There was a significant correlation between serum and hair level of iron (r = 0.504, P = 0.001), zinc (r = 0.684, P = 0.0001), and copper (r = 0.759, P = 0.0001) in patients and controls. Discussion and Conclusion. According to this study, there was no statistically significant difference between trace elements among AA patients and controls. So the trace elements level in hair and serum may not be relevant to the immunologic dysfunction that exists in AA patients.

CCR4 C1014T and CCL22 C16A genetic variations in the Iranian patients with colorectal adenocarcinoma.

C-C motif chemokine 22 (CCL22) C16A genetic variation (rs4359426) and C-C chemokine receptor type 4 (CCR4) C1014T variation (rs2228428) have been suggested to affect the expression level of the cognate proteins. Here we tried to investigate the plausible association of these polymorphisms with development of colorectal cancer. 165 patients with colorectal adenocarcinoma (age 54.4±13.4) and 150 age- and sex-matched healthy individuals were enrolled. Genotyping was performed by PCR-RFLP methods. Results indicated the frequency of 16A allele in CCL22 gene to be 31/330(9.4%) and 33/300(11%) in patients and controls, respectively (p=0.59). The frequencies of CC, CA, and AA genotypes at this locus were not significantly different between patients and controls (135/165; 81.8%, 29/165; 17.6%, 1/165; 0.6% in the patients and 121/150; 80.1%, 25/150; 16.6% and 4/150; 2.6% in the control group, p= 0.34). At the locus 1014 in CCR4, T allele was observed with the frequency of 107/330 (32.4%) and 83/300 (27.7%) in patients and controls, respectively (p=0.22). Analyses indicated no significant differences in the frequencies of CC, CT and TT genotypes at this locus between patients and controls (77/165; 46.7%, 69/165; 41.8% and 19/165; 11.5%; versus 83/150; 55.0%, 51/150; 33.8% and 16/150; 10.6%, respectively, p= 0.29). The presence of individual genotypes was not associated with clinicopathological characteristics of the disease, including tumor size, tumor grade and LN involvement (all with p<0.05). These findings collectively suggested that CCR4 C1014T and CCL22 C16A genetic variations were neither associated with the risk, nor with the progression of colorectal cancer in Iranian population.

Effects of T208E activating mutation on MARK2 protein structure and dynamics: Modeling and simulation.

Microtubule Affinity-Regulating Kinase 2 (MARK2) protein has a substantial role in regulation of vital cellular processes like induction of polarity, regulation of cell junctions, cytoskeleton structure and cell differentiation. The abnormal function of this protein has been associated with a number of pathological conditions like Alzheimer disease, autism, several carcinomas and development of virulent effects of Helicobacter pylorißßαßßß.

Adipose Tissue Derived Multipotent Mesenchymal Stromal Cells Can Be Isolated Using Serum-free Media.

BACKGROUND: Mesenchymal stromal cells (MSCs) as multipotent cells with the capacity to be differentiated into several cell lineages are promising sources for cell therapy and tissue engineering nowadays. Today most of culturing media are supplemented with fetal bovine serum (FBS). But FBS containing culturing media may raise the possibility of zoonotic infections and immunological reactions in cell therapy conditions. Numerous investigations have been performed to assess the use of FBS-free culturing systems for bone marrow derived mesenchymal stromal cell isolation. OBJECTIVES: The present investigation aimed to assess the effect of serum-free media on growth and differentiating capacity of adipose tissue- derived MSCs. MATERIALS AND METHODS: Approximately, 1cm3 surgically waste sterile adipose tissue was digested with collagenase-I leading to a single cell suspension. The isolated cells were cultured in Ultra Culture media supplemented with 2% Ultroser G. MSC's isolation was confirmed with respect to morphology, flowcytometry, adipogenic and osteogenic differentiation potentials. RESULTS: The isolated cells showed adherent spindle shaped morphology, expanded rapidly and revealed expected MSC flowcytometric characteristics; they were positive for CD73, CD90, CD105, CD44, CD166, CD44 and negative for hematopoietic antigen such as CD45, CD34 and CD14. They could also be differentiated successfully into osteoblast and adipocyte, being confirmed by using Alizarin Red and Oil red O staining, respectively. CONCLUSIONS: According to the results of the present study, it can be concluded that adipose derived MSCs can be cultured in serum-free media with no change in their differentiating capacity. This finding gives us a hope for future cell therapy studies and trials with little concern about zoonotic infections or immunological reaction.