The Protein Journal最新文献

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site’s intrinsic dynamic movement to estimate the new peptide’s role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.

Graphical Abstract

Protein conformation is affected by interaction of several small molecules resulting either stabilization or disruption depending on the nature of the molecules. In our earlier communication, Hg²⁺ was known to disrupt the native structure of α-Cgn A leading to aggregation (Ansari, N.K., Rais, A. & Naeem, A. Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A. Protein J (2024). https://doi.org/10.1007/s10930-024-10187-z). Accumulation of β-rich aggregates in the living system is found to be linked with copious number of disorders. Here, we have investigated the effect of varying concentration of doxorubicin (DOX) i.e. 0-100 µM on the preformed aggregates of α-Cgn A upon incubation with 120 µM Hg²⁺. The decrease in the intrinsic fluorescence and enzyme activity with respect to increase in the Hg²⁺ concentration substantiate the formation of aggregates. The DOX showed the dose dependent decrease in the ThT fluorescence, turbidity and RLS measurements endorsing the dissolution of aggregates which were consistent with red shift in ANS, confirming the breakdown of aggregates. The α-Cgn A has 30% α-helical content which decreases to 3% in presence of Hg²⁺. DOX increased the α-helicity to 28% confirming its anti-aggregatory potential. The SEM validates the formation of aggregates with Hg²⁺ and their dissolution upon incubation with the DOX. Hemolysis assay checked the cytotoxicity of α-Cgn A aggregates. Docking revealed that the DOX interacted Lys203, Cys201, Cys136, Ser159, Leu10, Trp207, Val137 and Thr134 of α-Cgn A through hydrophobic interactions and Gly133, Thr135 and Lys202 forms hydrogen bonds.

The interaction between vascular endothelial growth factor A (VEGFA) and VEGF receptor 1(VEGFR1) is a central focus for drug development in pathological angiogenesis, where aberrant angiogenesis underlies various anomalies necessitating therapeutic intervention. Identifying hotspots of these proteins is crucial for developing new therapeutics. Although machine learning techniques have succeeded significantly in prediction tasks, they struggle to pinpoint hotspots linked to angiogenic activity accurately. This study involves the collection of diverse VEGFA and VEGFR1 protein sequences from various species via the UniProt database. Electron-ion interaction Potential (EIIP) values were assigned to individual amino acids and transformed into frequency-domain representations using discrete Fast Fourier Transform (FFT). A consensus spectrum emerged by consolidating FFT data from multiple sequences, unveiling specific characteristic frequencies. Subsequently, the Stockwell Transform (ST) was employed to yield the hotspots. The Resonant Recognition Model (RRM) identified a characteristic frequency of 0.128007 with an associated wavelength of 1570 nm and RRM-ST identified hotspots for VEGFA (Human 36, 46, 48, 67, 71, 74, 82, 86, 89, 93) and VEGFR1 (Human 224, 259, 263, 290, 807, 841, 877, 881, 885, 892, 894, 909, 913, 1018, 1022, 1026, 1043). These findings were cross-validated by Hotspots Wizard 3.0 webserver and Protein Data Bank (PDB). The study proposes using a 1570 nm wavelength for photo bio modulation to boost VEGFA/VEGFR1 interaction in the condition that is needed. It also aims to reduce VEGFA/VEGFR2 interaction, limiting harmful angiogenesis in conditions like diabetic retinopathy. Also, the identified hotspots assist in designing agonistic or antagonistic peptides tailored to specific medical requirements with abnormal angiogenesis.

Plasmodium falciparum (P. falciparum), which causes the most severe form of malaria, if left untreated, has 24 h window in which it can cause severe illness and even death. The aim of this study was to create the most comprehensive and informative secretory-proteome possible by combining high-accuracy and high-sensitivity protein identification technology. In this study, we used Plasmodium falciparum 3D7 (Pf3D7) as the model parasite to develop a label-free quantification proteomic strategy with the main goal of identifying Pf3D7 proteins that are supposed to be secreted outside the infected erythrocytes in the spent media culture during the in-vitro study. The spent culture media supernatant was subjected to differential and ultra-centrifugation steps followed by total protein extraction, estimation, and in-solution digestion using trypsin, digested peptides were analyzed using Nano-LC coupled with ESI for MS/MS. MS/MS spectra were processed using Maxquant software (v2.1.4.0.). Non-infected erythrocytes incubated spent cultured media supernatant were considered as control. Out of discovered 38 proteins, proteins belonging to P. falciparum spp. were EGF-like protein (C0H544), Endoplasmic reticulum chaperone GRP170 (C0H5H0), Small GTP-binding protein sar1 (Q8I1S0), Erythrocyte membrane protein 1, PfEMP1 (Q8I639), aldehyde reductase (Q8ID61), Conserved Plasmodium proteins (Q8IEH3, Q8ILD1), Antigen 332, DBL-like protein (Q8IHN4), Fe-S cluster assembly protein (Q8II78), identified and chosen for further in-depth investigation. This study highlights the value of secretory Plasmodium proteins play crucial roles in various aspects of the disease progression and host-pathogen interactions which can serve as diagnostic markers for malaria infection.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × ₁::CCDC124, GFP11 × ₄::CCDC124, or CCDC124::GFP11 × ₄). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × _{1(or X4)}::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11_× 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × ₄::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144–159; 164; 171–183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

In the realm of parasitology, autophagy has emerged as a critical focal point, particularly in combating Leishmaniasis. Central to this endeavour is the recognition of the protein ATG8 as pivotal for the survival and infectivity of the parasitic organism Leishmania major, thereby making it a potential target for therapeutic intervention. Consequently, there is a pressing need to delve into the structural characteristics of ATG8 to facilitate the design of effective drugs. In this study, our efforts centered on the purification of ATG8 from Leishmania major, which enabled novel insights into its structural features through meticulous spectroscopic analysis. We aimed to comprehensively assess the stability and behaviour of ATG8 in the presence of various denaturants, including urea, guanidinium chloride, and SDS-based chemicals. Methodically, our approach included secondary structural analysis utilizing CD spectroscopy, which not only validated but also augmented computationally predicted structures of ATG8 reported in previous investigations. Remarkably, our findings unveiled that the purified ATG8 protein retained its folded conformation, exhibiting the anticipated secondary structure. Moreover, our exploration extended to the influence of lipids on ATG8 stability, yielding intriguing revelations. We uncovered a nuanced perspective suggesting that targeting both the lipid composition of Leishmania major and ATG8 could offer a promising strategy for future therapeutic approaches in combating leishmaniasis. Collectively, our study underscores the importance of understanding the structural intricacies of ATG8 in driving advancements towards the development of targeted therapies against Leishmaniasis, thereby providing a foundation for future investigations in this field.

Graphical Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent cancer types in the world and accounts for the majority of cases of primary liver cancer. A crucial part of the carcinogenesis of HCC involves aberrant stimulation of the FGF19-FGFR4 signaling pathway. Therefore, FGFR4 inhibition has become a strategic therapeutic approach for the treatment of HCC. However, the clinical treatment procedure is significantly hampered by the prevalence of kinase inhibitors resistance. It was recently established that the activation of EGFR signaling was found to be one of the primary mechanisms mediating the acquired resistance to FGFR4 inhibitors, moreover, sensitivity to FGFR4 inhibitors was effectively restored by inhibiting EGFR. These results provide compelling evidence that dual inhibition of EGFR and FGFR4 could represent a viable therapeutic approach to overcome resistance, hence enhanced management of HCC. To this end, we proposed a dual irreversible inhibition strategy through covalent binding by naturally occurring electrophilic warhead-bearing compounds (curcumin, deoxyelephantopin, eupalmerin acetate, syringolin A and andrographolide) to covalently target both EGFR and FGFR4 through cysteine residues, Cys797 and Cys552, respectively. Covalent docking and covalent molecular dynamics (MM/MD^cov) simulations combined with thermodynamic binding free energy calculations were performed, and the results were compared against known potent and selective covalent EGFR and FGFR4 inhibitors with available X-ray crystal structures, Afatinib and BLU9931, respectively. Curcumin, deoxyelephantopin, eupalmerin acetate, syringolin A, and andrographolide showed relative binding free energies of -22.85, -17.14, -12.98, -21.81, and − 19.00 kcal/mol against EGFR and − 41.06, -29.45, -24.76, -40.11, and − 37.55 kcal/mol against FGFR4, respectively. The mechanisms of binding were emphasized by hydrogen bonding and binding forces analysis as well as active site physicochemical profiling. The findings of this study identified that curcumin, syringolin A and andrographolide—but not eupalmerin acetate or deoxyelephantopin —could be viable dual EGFR and FGFR4 covalent irreversible inhibitors and could be implemented in HCC combination therapy protocols alone or in conjunction with other chemotherapeutic agents. Investigations of this study conclusively indicate dual blockade of EGFR and FGFR4 may be a promising future therapeutic strategy for enhanced management of HCC.

Determining the physicochemical properties of a protein can reveal important insights in their structure, biological functions, stability, and interactions with other molecules. Although tools for computing properties of proteins already existed, we could not find a comprehensive tool that enables the calculations of multiple properties for multiple input proteins on the proteome level at once. Facing this limitation, we developed Multiple Protein Profiler (MPP) 1.0 as an integrated tool that allows the profiling of 12 individual properties of multiple proteins in a significant manner. MPP provides a tabular and graphic visualization of properties of multiple proteins. The tool is freely accessible at https://mproteinprofiler.microbiologyandimmunology.dal.ca/.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg²⁺ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg²⁺. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg²⁺, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg²⁺ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg²⁺. Conclusively, the presence of Mg²⁺ induces a conformation in KpPrAT that favours nucleotide binding.