Gly Residues Research Articles

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature TLLPS. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e., single-phase) and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich “droplets”. Comparison of the experimental results with simulations of the sensitivity of 2D ssNMR crosspeaks to changes in populations of β-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%.

Photochemical and Collision-Induced Cross-Linking in Stereochemically Distinct Scaffolds of Peptides and Nitrile Imines in Gas-Phase Ions.

Intramolecular cross-linking between peptides and nitrile-imine intermediates was studied in stereochemically distinct conjugates in which the reacting components were mounted on cis-1,2-cyclohexane and trans-1,4-cyclohexane scaffolds that we call 1,2-s-peptides and 1,4-s-peptides, respectively. The nitrile-imine intermediates were generated by N2 loss from 2,5-diaryltetrazole tags upon UV-photodissociation at 213 and 250 nm or by collision-induced dissociation, and further interrogated by CID and UVPD-MS3. Peptide fragment ion series originating from linear structures and macrocyclic cross-links were distinguished and used to quantify the cross-linking yields. The yields in MS2 varied between 27% for AAAG conjugates to 78% for GAAAK conjugates, depending on the peptide sequence. The CID-MS3 yields were in a 57-97% range, depending on the peptide sequence. Structures of 1,2-s-peptide and 1,4-s-peptide ions as well as several of their nitrile-imine intermediates and cross-links were investigated by high-resolution cyclic ion mobility in combination with Born-Oppenheimer molecular dynamics and density functional theory calculations. Matches between the experimental and calculated collision cross sections and ion relative Gibbs energies were used to assign peptide structures. Peptide conjugates C-terminated with Gly and Lys residues underwent cross-linking by the carboxyl group, as established by MS3 sequencing and corroborated by carboxyl blocking experiments that lowered the cross-linking yields. Peptide conjugates C-terminated with Arg also cross-linked via the side-chain guanidine group. A notable feature of the 1,4-s-peptide ions was the participation of low-energy twist-boat cyclohexane conformers that was enforced by strong hydrogen bonds between the peptide and nitrile imine.

Biophysical and structural analyses of the interaction between the SHANK1 PDZ domain and an internal SLiM.

The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

A phase I dose escalation trial of BCD-145 in patients with unresectable or metastatic melanoma.

9526 Background: BCD-145 (nurulimab) is a fully human IgG1 mAb with modified Fc-fragment (the 1st Gln is replaced by Glu in the heavy chain sequence, and there is no terminal dipeptide composed by Gly and Lys residues) that binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and blocks the interaction between CTLA-4 and its ligands. This allows to stimulate T-cell proliferation, cytokine production and induces antitumor immune response. Here we present the results of multicenter open-label single-arm multi-cohort phase I trial of pharmacokinetics (PK), pharmacodynamics (PD), safety, and immunogenicity of BCD-145 in pts with unresectable/metastatic melanoma (unr/mM). Methods: BCD-145 monotherapy in 3+3 dose escalation [0.1, 0.3, 1, 3, 10 mg/kg intravenously Q3W] was given to 15 pts with unr/mM with measurable disease, who had failed prior therapy, ECOG 0-2. Pts with brain mts, previous therapy with aCTLA-4 and/or aPD-1/PD-L1 drugs were not allowed. The main objective of the study was to evaluate PK, PD, tolerability, immunogenicity, safety, dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) identification. Secondary objectives included tumor response rate (irRECIST). Results: BCD-145 proved safety and overall was well tolerated. DLT and MTD were not reached. Any grade (Gr) adverse events (AEs) occurred in 100% (15/15) of pts. 33.3% (5/15) of pts had AE/SAE of Gr 3-4, including hypertension Gr 3 (1 at 1 mg/kg), hypercalcemia Gr 4 (2 at 1 mg/kg), hypokalemia Gr 3 (2 at 1 mg/kg), neutropenia Gr 3 (1 at 0.3 mg/kg), anemia Gr 3 (1 at 1 mg/kg), lung infection Gr 3 with hospitalization (1 at 1 mg/kg). Only 2 pts had a treatment-related AE Gr 3-4. Immuno-related AE were identified in 46.7% (7/15), manifested by symptoms of thyroiditis (mainly Gr 1) and diarrhea Gr 2 (1 at 10 mg/kg). There was 1 death (at 1 mg/kg) due to progression. There is dose-dependent peak study drug serum concentrations and systemic exposure. Two dosage regimens (1 mg/kg, 3 mg/kg Q3W) allowed to achieve the optimal concentration. BCD-145 increased subpopulations of activated HLA-DR+ CD4+ and CD8+ T lymphocytes, CD4+ICOS+ T-lymphocytes, absolute lymphocyte counts in all cohorts without significant differences. Binding and neutralizing Abs against BCD-145 were detected in 2 pts from different dose cohorts (0.3 mg/kg, 1 mg/kg). PR and SD were registered in 2 pts of each response group. 30.8% of pts achieved disease control, which is consistent with published data on other CTLA-4 inhibitors. A dose of 1 mg/kg is determined as recommended phase 2 dose (RP2D). Conclusions: BCD-145 demonstrated a tolerable safety profile and preliminary anti-tumor activity in pts with unr/mM. Anti-tumor effects of BCD-145 were observed in pts who had progressed on prior treatment. Further co-targeting PD-1 and CTLA-4 simultaneously will achieve a better anti-tumor activity due to cooperative binding to exhausted T-cell subsets. Clinical trial information: NCT03472027 .

Crystal structure of a newly identified M61 family aminopeptidase with broad substrate specificity that is solely responsible for recycling acidic amino acids.

Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP-dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure-function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3-7 amino acids. Further, we report the high-resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin-bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild-type and m61xc-knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N-termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post-proteasomal protein degradation in prokaryotes.

NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

Adenosine triphosphate alleviates high temperature-enhanced glyphosate toxicity in maize seedlings

Extracellular ATP plays a key role in regulating plants stress responses. Here, we aimed to determine whether ATP can alleviate the glyphosate toxicity in maize seedlings under high temperature by regulating antioxidant responses. Foliar spraying with 100 μM glyphosate inhibited the growth of maize seedlings at room temperature (25 °C), leading to an increase in shikimic acid accumulation and oxidative stress (evaluated via lipid peroxidation, free proline, and H2O2 content) in the leaves, all of which were further exacerbated by high temperature (35 °C). The growth inhibition and oxidative stress caused by glyphosate were both alleviated by exogenous ATP. Moreover, the glyphosate-induced antioxidant enzyme activity and antioxidant accumulation were attenuated by high temperature, while ATP treatment reversed this inhibitory effect. Similarly, qPCR data showed that the relative expression levels of antioxidant enzyme-related genes (CAT1, GR1, and γ-ECS) in maize leaves were upregulated by ATP before exposure to GLY. Moreover, high temperature-enhanced GLY residue accumulation in maize leaves was reduced by ATP. ATP-induced detoxification was attenuated through NADPH oxidase (NOX) inhibition. Higher NOX activities and O2•− production were noted in ATP-treated maize leaves compared to controls prior to GLY treatment, indicating that the extracellular ATP-induced alleviation of GLY toxicity was closely associated with NOX-dependent reactive oxygen species signalling. The current findings present a new approach for reducing herbicide toxicity in crops exposed to high temperatures.

Myristoylated GRASP55 dimerizes in the presence of model membranes

The Golgi Reassembly and Stacking Proteins (GRASPs) are engaged in various functions within the cell, both in unconventional secretion mechanisms and structuring and organizing the Golgi apparatus. Understanding their specific role in each situation still requires more structural and functional data at the molecular level. GRASP55 is one of the GRASP members in mammals, anchored to the membrane via the myristoylation of a Gly residue at its N-terminus. Therefore, co-translational modifications, such as myristoylation, are fundamental when considering a strategy to obtain detailed information on the interactions between GRASP55 and membranes. Despite its functional relevance, the N-terminal myristoylation has been underappreciated in the studies reported to date, compromising the previously proposed models for GRASP-membrane interactions. Here, we investigated the synergy between the presence of the membrane and the formation of oligomeric structures of myristoylated GRASP55, using a series of biophysical techniques to perform the structural characterization of the lipidated GRASP55 and its interaction with biological lipid model membranes. Our data fulfill an unexplored gap: the adequate evaluation of the presence of lipidations and lipid membranes on the structure-function dyad of GRASPs. Communicated by Ramaswamy H. Sarma

Collagen derived Gly-Pro-type DPP-IV inhibitory peptides: Structure-activity relationship, inhibition kinetics and inhibition mechanism

Our previous study has demonstrated that both the amino acid at N3 position and peptide length affected the DPP-IV inhibitory activity of Gly-Pro-type peptides. To further elucidate their molecular mechanism, a combined approach of QSAR modeling, enzymatic kinetics and molecular docking was used. Results showed that the QSAR models of Gly-Pro-type tripeptides and Gly-Pro-type peptides containing 3–12 residues were successfully constructed by 5z-scale descriptor with R2 of 0.830 and 0.797, respectively. The lower values of electrophilicity, polarity, and side-chain bulk of amino acid at N3 position caused higher DPP-IV inhibitory activity of Gly-Pro-type peptides. Moreover, an appropriate increase in the length of Gly-Pro-type peptides did not change their competitive inhibition mode, but decreased their inhibition constants (Ki values) and increased interactions with DPP-IV. More importantly, the interactions between the residues at C-terminal of Gly-Pro-type peptides containing 5 ∼ 6 residues with S2 extensive subsites (Ser209, Phe357, Arg358) of DPP-IV increased the interactions of Gly residue at N1 position with the S2 subsites (Glu205, Glu206, Asn710, Arg125, Tyr662) and decreased the acylation level of DPP-IV-peptide complex, and thereby increasing peptides’ DPP-IV inhibitory activity.

NAD<sup>+</sup>-dependent format dehydrogenase from the thermotolerant yeast <i>ogataea parapolymorpha</i>: properties and protein engineering of the <i>n</i>-terminal sequence

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. The recombinant enzyme with an additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in an active and soluble form with a yield of more than 1 g per liter of medium. In the present work, a detailed comparison of this enzyme with FDH from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate the effect of the N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that the addition of an Ala residue to the N-terminus reduces the rate constant of thermal inactivation four times compared with the addition of a Gly residue. The addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to an acceleration of purification, practically does not affect the kinetic parameters, but somewhat reduces the temperature stability, which, however, can be restored to the level of OpaFDH_AK by adding 0.5 M NaCl.

Heterobivalent Peptide Agonists Comprising Covalently Linked Protease Activated Receptor 1 and Protease Activated Receptor 3 Peptides Mimic the Cytoprotective Cell Signaling Activities of Activated Protein C

Introduction: Activated protein C (APC) exhibits both anticoagulant and cytoprotective cell signaling actions, including anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Extensive in vitro and in vivo data for APC's cell signaling benefits identified requirements for two G-protein coupled receptors (GPCR), protease-activated receptor (PAR) PAR1 and PAR3 which are cleaved by APC at the noncanonical sites (R46 and R41, respectively). Agonism of PARs following proteolysis of the N-terminal tail is driven by the newly revealed N-terminus and related synthetic peptides, e.g., for APC, PAR1 peptides (P1) beginning with N47 or PAR3 peptides (P3) beginning with G42. We found that the non-canonical P1-47-66 and P3-42-65 peptides mimic APC in that each is anti-inflammatory by reducing NLRP3-driven caspase-1 activity in human THP-1 cells (Healy LD et al, J Thromb Haemost 2021;19:269-280). Remarkably, these two peptides act synergistically to attenuate activity of caspase-1 at low concentrations where either peptide alone has little activity. Thus, we undertook studies of P1 peptides that were covalently linked to P3 peptides for ability to mimic APC's anti-inflammatory and endothelial barrier stabilization actions. Methods: Monocytes (THP-1 cells) were differentiated into macrophages using PMA, then pretreated with APC or peptides prior to stimulation with lipopolysaccharide and adenosine triphosphate to induce caspase-1 whose activity was measured using a luminescent caspase-1 substrate. Endothelial barrier stability was monitored for transendothelial electrical resistance (TEER) using an electric cell-substrate sensing system (iCelligence apparatus) when cultured human EAhy926 or murine aortic endothelial cells (EC) were exposed to thrombin treatment (0.25 nM) plus or minus treatment with P1 and/or P3 peptides or G10-related peptides. Synthetic P1 and P3 sequence-derived peptides of various compositions were made, and bivalent P1:P3 peptides were made using oligo-Gly-peptide linkers containing from 4 to 14 Gly residues. Results: Using THP-1 cell anti-inflammatory assays for various peptides, studies showed that P1-47-55 was as potent as P1-47-66 and that the N-terminal N47 was essential. In anti-inflammatory activity assays, some P3 peptides shorter than P3-42-65 (e.g., P3-44-54, P3-51-65, and P3-51-59) were as active as P3-42-65. Remarkably, this is the first example for any PAR-agonist peptide that does not require a native-like sequence with an intact free alpha-amino N-terminus to agonize the PAR response. For bivalent P1:P3 studies, we covalently linked P3-47-55 to P3-51-65 using oligo-Gly linkers with 4-14 Gly residues. In anti-inflammatory activity assays, the maximal activity was seen for a 10-Gly linker. The bivalent peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65, designated as the G10 peptide, was much more potent than any P1 peptide or P3 peptide in anti-inflammatory THP-1 cell assays in dose-response studies. In TEER studies of thrombin-challenged EAhy926 EC where APC markedly attenuates thrombin-induced endothelial barrier dysfunction, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was much more potent than the P1-47-55 peptide or any combination of P1-47-55 with a P3 peptide. The P3 peptides alone were very weak. In TEER studies of thrombin-challenged murine aortic EC where murine APC markedly attenuates thrombin-induced endothelial barrier dysfunction, the G10 peptide comprising murine PAR sequences mimicked murine APC's protective actions. Conclusions: Covalent linkage of a PAR1-derived peptide agonist to a PAR3-derived peptide agonist using a 10-Gly-residue linker provided the first heterobivalent PAR peptide GPCR agonist. This heterobivalent G10 peptide potently mimicked APC's cytoprotective activities of anti-inflammation and endothelial barrier stabilization. Molecular mechanisms for G10 likely involve both orthosteric and allosteric effects for PAR1 and PAR3 which can form heterodimers. Given APC's beneficial cell signaling effects in many preclinical injury models and given the increasing usage of peptide-based drugs, studies of the G10 peptide for beneficial APC-mimicking effects may help to identify important directions for future translational research.

Docking and MM study of non-structural protein (NS5) of Japanese Encephalitis Virus (JEV) with some derivatives of adenosyl

Introduction: The flavivirus NS5, a non-structural protein of Japanese Encephalitis Virus (JEV), a serious deadly human pathogen responsible for epidemics in South East Asia, consists of N-terminal methyl transferase (MTase) domain and RNA-dependent RNA polymerase (RdRp) is known for unique viral genome replication and cap formation activity. S-adenosyl executes a crucial function in these viral activities. S-adenosyl derivatives are chosen as potential binders with the MTase domain of NS5 based on MM and docking studies.Methods: MM GBSA (Generalized Born Surface Area) simulation were performed to evaluate the binding energy, following the 100 nanosecond (ns) production MD simulation in the periodic boundary condition (PBC) for the selected docked ligands with NS5. Quasi-harmonic entropy of the ligands was also calculated with semi-empirical calculations at the PM3/PM6 level supporting docking and MM-GBSA results.Results and discussion: The residue-wise decomposition energy reveals that the key hydrophobic residues Gly 81, Phe 133, and Ile 147 in the RdRp-MTase interface, indicate the biological relevance. These residues act as the key residue stabilizer, binding vigorously with S-Adenosyl derivatives in the vicinity of the interface between the MTase domain and RdRp. This paves the way for the other potential drug as an inhibitor for the enzymatic activity of the NS5.

A common pathway for detergent-assisted oligomerization of Aβ42

Amyloid beta (Aβ) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aβ42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aβ42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aβ42 tetramer. Here we investigate the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS- and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel β-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous Aβ aggregation at cellular interfaces.

Arg389Gly beta-1-adrenergic receptor polymorphism determines neutrophil-stunning-associated cardioprotection by metoprolol

Abstract Background Cardioprotective strategies to limit infarct size (IS) have become crucial. The damaged inflicted on the myocardium is the result of the processes of Ischemia and Reperfusion (I/R). In the clinical trial METOCARD-CNIC, it was shown that the intravenous (i.v.) pre-reperfusion administration of the β1-selective antagonist metoprolol reduces IS and improves long-term cardiac function in patients after acute myocardial infarction (1). However, the small sample size precludes a definite conclusion. Despite not having demonstrated a solid clinical benefit in terms of hard endpoints reduction, the clinical guidelines for the management of ST-Elevation Myocardial Infarction (STEMI) recommend the early administration of i.v. β-blockers in patients undergoing Primary Coronary Intervention. Additionally, our group has recently described that the beneficial effect of metoprolol against myocardial I/R injury is due to neutrophil stunning (2), which represents a one-of-a-kind property not shared by other i.v. β-blockers, such as atenolol or propranolol (3). Purpose Since β1-adrenergic receptor (ADRB1) is one of major targets of pharmacological therapy, great efforts are now underway to account for the relevance of the polymorphic variation. Here we explore whether the cardioprotection exerted by metoprolol is dependent on genetic polymorphisms in human ADRB1. Methods Patients included in the METOCARD-CNIC trial were grouped into the different variants for the ADRB1, rs1801252 and rs1801253 (SerGly49 and Arg389Gly, respectively), which might affect their response to the beta-1 modulation exerted by metoprolol on neutrophils and thus its cardioprotective effect. Additionally, the polymorphic-dependent effect of metoprolol on neutrophil migration was evaluated in healthy volunteers, whose neutrophils were exposed across the transwell filter to the CXCL1 in the presence or absence of metoprolol. Drug-receptor interaction properties were evaluated in silico. Results We found that Arg389Gly polymorphism of the human ADRB1 impacts the cardioprotective effect of metoprolol in STEMI patients. Therefore, Arg389 homozygosity was associated with lower infarcts (Figure 1) and better long-term left ventricle ejection fraction when metoprolol was intravenously administered prior reperfusion. Moreover, the disrupting effect of metoprolol on neutrophil migration was only observed in Arg389 homozygote volunteers (Figure 2). These differences were attributed to alterations in the drug-receptor complex stability when Gly residues are present in position 389 of the ADRB1. Conclusions From our perspective, these results are the first showing a polymorphic implication in drug response against myocardial I/R injury. All the data presented refine cardiovascular pharmacotherapy, and have major implications for the prospective clinical trial design aiming at positioning metoprolol as the beta-blocker of choice to reduce hard endpoints in STEMI patients.CardioprotectionNeutrophil migration

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster.

Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.

NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence.

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.

Proteomics analysis of the influence of proteolysis on the subsequent glycation of myofibrillar protein

Proteomics was used to study the influence of proteolysis on the glycation of myofibrillar proteins (MPs). Proteolysis by papain and proteinase K generated the highest level of amino acids (AAs) and peptides, respectively. Both the glycation degree (A value increased from 0.173 to 0.202-0.348) and speed (k value increased from 0.0099 to 0.0132-0.0145) were enhanced by proteolysis using papain and proteinase K. Proteomics analysis revealed that proteolysis largely enhanced the glycation site number in Lys, Arg and N-terminal residues (eg. Leu, Gly, Thr, Ala, Met, Ile, Phe and Val residues in myosin light chain). Proteolysis by papain preferentially acted on actin and therefore specifically increased the glycation sites from actin. Proteolysis reduced the level of aldehydes but enhanced the aromatic E-nose signals, possibly due to the combination of aldehydes with released AAs/peptides. The proteomics analysis helped to detail the relationship between proteolysis and subsequent glycation/flavour formation.

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.