Changes In Secondary Structure Research Articles

Unraveling the Role of BRCA1 variants in Dysregulation of Transcriptional and Post-Transcriptional Mechanisms in Breast Cancer

Objective: To screen BRCA1 gene variants and predict potential role of the identified variants in breast cancer. Method: This case-control study included two hundred and fifty breast cancer patients and equal healthy individuals from the Federal Breast Cancer Screening Centre, Pakistan Institute of Medical Sciences, Islamabad from March 2021- January 2023. Demographic data was collected through questionnaires and clinical data was assessed using mammograms, ultrasound, histopathology and immunohistochemistry reports. Polymerase chain reaction and Sanger sequencing approach were used to detect variants in BRCA1 gene. In-silico analyses were carried out to predict mutation effect, miRNA binding site alterations and change in mRNA structure and stability. Results: Invasive ductal carcinoma was the most prevalent type of breast cancer. Old age [OR: 2.8149 (1.5995 to 4.9538) p value = 0.0003] and family history [OR: 4.3186 (1.7336 to 10.7581) p value = 0.001] were significant breast cancer risk. Six variants were identified. Two novel missense variants, Chr17:43082553A>T and Chr17:43093710A>T were predicted deleterious as these disrupted interaction with PALB2 and importin alpha’s NLS2 site, respectively. In silico analysis predicted the loss of hsa-miR-1179 binding site due to variant Chr17:43093220T>C. Moreover, four variants were predicted to affect the mRNA structure and stability. Conclusion: Two novel variants were predicted to be pathogenic. In-silico analysis predicted the loss of miRNA binding site along with change in mRNA secondary structure plus stability, possible mechanisms for oncogenesis. Further, expressional studies are required to confirm BRCA1 gene dysregulation in breast cancer due to these variants. doi: https://doi.org/10.12669/pjms.40.6.8761 How to cite this: Majeed AI, Zinan S, Faryal R. Unraveling the Role of BRCA1 variants in Dysregulation of Transcriptional and Post-Transcriptional Mechanisms in Breast Cancer. Pak J Med Sci. 2024;40(6):1093-1098. doi: https://doi.org/10.12669/pjms.40.6.8761 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pea protein-inulin conjugate prepared by atmospheric pressure plasma jet combined with glycosylation: structure and emulsifying properties

Pea protein is one of plant proteins with high nutritional value, but its lower solubility and poor emulsifying properties limit its application in food industry. Based on wet-heating glycosylation of pea protein and inulin, effects of discharge power of atmospheric pressure plasma jet (APPJ) on structure, solubility, and emulsifying ability of pea protein-inulin glycosylation conjugate were explored. Results indicated that the APPJ discharge power did not affect the primary structure of pea protein. However, changes in secondary and spatial structure of pea protein were observed. When APPJ discharge power was 600 W, the solubility of glycosylation conjugate was 75.0% and the emulsifying stability index was 98.9 min, which increased by 14.85 and 21.95% than that of only glycosylation sample, respectively. These findings could provide technical support for APPJ treatment combination with glycosylation to enhance the physicochemical properties of plant-based proteins.

In vitro susceptibility of grass carp fish (Ctenopharyngodon idella) to oxidized myosin on structural change of protein secondary structure associated with reactive oxygen species (ROS) treatment

This study aimed to investigate the in vitro susceptibility of grass carp (Ctenopharyngodon idella) oxidized myosin, with the treatment of H2O2 levels being 0, 25, 50, and 100 μmol/L, respectively. The results showed that in vitro oxidation of myosin, reactive oxidative species promoted protein modifications that propagated the conformation changes on myosin secondary structure, leading to a significant increase (P < 0.05) in the protein carbonyl content in the high oxidation treatment, when compared to the low and medium treatments. The results of the Western blot analysis and sodium-polyacrylamide amide-dodecyl sulfate gel electrophoresis demonstrated that in vitro oxidation actively promoted the degradation of protein molecular weight and Desmin (53 kDa) deterioration in the sample with the highest concentration treatment of H2O2. Circular dichroism spectra showed a decrease in α-helix content from 70.1% to 29.8% and an increase in β-sheet percentage from 5.6% to 17.4%. Meanwhile, cysteine and methionine as the free amino acids were most susceptible to oxidative change due to their high sensitivity to the reaction of the sulfur group of amino acids. Protein degradation induced by oxidation resulted in significant changes to the protein secondary structure, as evidenced by the observation of oxidized myosin under different H2O2 levels.

Synergistic coupling of flocculation and fenton reaction for enhanced sludge dewatering: Pivotal role of bi-functional cationic tannin

The challenges associated with sludge dewatering are primarily attributed to the rigid structure formed through the combination of sludge extracellular polymeric substances (EPS) and bound water. Although Fenton oxidation can effectively break down sludge EPS, it faces several challenges including the tough reduction of Fe3+ and the need for large quantities of reagents. In this study, we prepared a cationic tannin (CT) that performed well in flocculation, while also serving as an effective Fe3+ reducing agent due to its phenolic hydroxyl structure. A novel synergistic process coupling CT and the Fenton reaction, integrating flocculation and oxidation, was established for sludge conditioning and dehydration. This system exhibited superior sludge dewatering performance compared to the individual addition of CT or Fenton oxidation under similar conditions. The specific resistance filtration (SRF) of sludge decreased to 22 × 1011m/kg, and the moisture content of the sludge cake decreased to 63.7%. The observed compositional and secondary structural changes in each layer of the sludge’s EPS further confirm the enhanced efficiency of EPS breakdown and bound water release. Following the conditioning process, the sludge filtrate and mud cake met the necessary discharge requirements and could be further treated and utilized as valuable resources in subsequent processes. Additionally, the cost of the system was obviously lower than that of conventional Fenton reaction. This study proposes the use of CT as a bi-functional material, possessing both flocculation and reduction capabilities. By combining CT with the Fenton reaction, we developed an economical and efficient deep sludge dewatering technology.

Using Gaussian accelerated molecular dynamics combined with Markov state models to explore the mechanism of action of new oral inhibitors on Complex I

In this study, our focus was on investigating H-1,2,3-triazole derivative HP661 as a novel and highly efficient oral OXPHOS inhibitor, with its molecular-level inhibitory mechanism not yet fully understood. We selected the ND1, NDUFS2, and NDUFS7 subunits of Mitochondrial Complex I as the receptor proteins and established three systems for comparative analysis: protein-IACS-010759, protein-lead compound 10, and protein-HP661. Through extensive analysis involving 500 ns Gaussian molecular dynamics simulations, we gained insights into these systems. Additionally, we constructed a Markov State Models to examine changes in secondary structures during the motion processes.The research findings suggest that the inhibitor HP661 enhances the extensibility and hydrophilicity of the receptor protein. Furthermore, HP661 induces the unwinding of the α-helical structure in the region of residues 726-730. Notably, key roles were identified for Met37, Phe53, and Pro212 in the binding of various inhibitors. In conclusion, we delved into the potential molecular mechanisms of triazole derivative HP661 in inhibiting Complex I. These research outcomes provide crucial information for a deeper understanding of the mechanisms underlying OXPHOS inhibition, offering valuable theoretical support for drug development and disease treatment design.

Interaction between ammonium based ionic liquids and CRABP I Protein: A microenvironmental spectroscopic analysis

In this article, we have used circular dichroism (CD) and fluorescence to study the physicochemical behaviour of a β-sheet protein, CRABP I, in the presence of ammonium-based ionic liquids (ILs) as a co-solvent. From fluorescence spectroscopy, a rise in intensity was observed in the presence of ionic liquids in the micromolar range, but a reversal in fluorescence intensity at higher concentrations (millimolar range). The microenvironment of aromatic amino acids, tyrosine (Tyr) and tryptophan (Trp), have been compared using synchronous fluorescence data, and the results demonstrate a similar trend in both cases as it has been seen in steady-state spectra. By using CD spectroscopy to examine conformational behaviour, we found that the protein's secondary structure hardly changes at all. Functionality test showed marginal changes in secondary structure of the protein in the presence of ionic liquids. Binding study revealed the contribution of van der waal’s interaction in the case of N2, N4 and N6 and hydrophobic force in the case of N8. The computational study demonstrated that these biophysical behaviours can be accounted for by the hydrophobic interactions between proteins and ionic liquids.

Elucidating the structural basis of vitamin B12 derivatives as novel potent inhibitors of PTP1B: Insights from inhibitory mechanisms using Gaussian accelerated molecular dynamics (GaMD) and in vitro study

Vitamin B12 is a group of biologically active cobalamin compounds. In this study, we investigated the inhibitory effects of methylcobalamin (MeCbl) and hydroxocobalamin acetate (OHCbl Acetate) on protein tyrosine phosphatase 1B (PTP1B). MeCbl and OHCbl Acetate exhibited an IC50 of approximately 58.390 ± 2.811 μM and 8.998 ± 0.587 μM, respectively. The Ki values of MeCbl and OHCbl Acetate were 25.01 μM and 4.04 μM respectively. To elucidate the inhibition mechanism, we conducted a 500 ns Gaussian accelerated molecular dynamics (GaMD) simulation. Utilizing PCA and tICA, we constructed Markov state models (MSM) to examine secondary structure changes during motion. Our findings revealed that the α-helix at residues 37–42 remained the most stable in the PTP1B-OHCbl Acetate system. Furthermore, upon binding of OHCbl Acetate or MeCbl, the WPD loop of PTP1B moved inward to the active pocket, forming a closed conformation and potentially obstructs substrate entry. Protein-ligand interaction analysis and MM-PBSA showed that OHCbl Acetate exhibited lower binding free energy and engaged in more residue interactions with PTP1B. In summary, our study confirmed the substantial inhibitory activity of OHCbl Acetate against PTP1B, with its inhibitory potency notably surpassing that of MeCbl. We demonstrated potential molecular mechanisms of OHCbl Acetate inhibiting PTP1B.

Raman spectroscopy in the study of amyloid formation and phase separation.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, β-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties

Contaminating microplastics can interact with food proteins in the food matrix and during digestion. This study investigated adsorption of chicken egg protein ovalbumin to polystyrene (PS, 110 and 260 μm) and polyethylene terephthalate (PET, 140 μm) MPs in acidic and neutral conditions and alterations in ovalbumin structure. Ovalbumin adsorption affinity depended on MPs size (smaller > larger), type (PS > PET) and pH (pH 3 > pH 7). In bulk solution, MPs does not change ovalbumin secondary structure significantly, but induces loosening (at pH 3) and tightening (at pH 7) of tertiary structure. Formed soft corona exclusively consists of full length non-native ovalbumin, while in hard corona also shorter ovalbumin fragments were found. At pH 7 soft corona ovalbumin has rearranged but still preserved level of ordered secondary structure, resulting in preserved thermostability and proteolytic stability, but decreased ability to form fibrils upon heating. Secondary structure changes in soft corona resemble changes in native ovalbumin induced by heat treatment (80 °C). Ovalbumin is abundantly present in corona around microplastics also in the presence of other egg white proteins. These results imply that microplastics contaminating food may bind and change structure and functional properties of the main egg white protein.

Radiolysis of myoglobin concentrated gels by protons: specific changes in secondary structure and production of carbon monoxide

While particle therapy has been used for decades for cancer treatment, there is still a lack of information on the molecular mechanisms of biomolecules radiolysis by accelerated ions. Here, we examine the effects of accelerated protons on highly concentrated native myoglobin, by means of Fourier transform infrared and UV–Visible spectroscopies. Upon irradiation, the secondary structure of the protein is drastically modified, from mostly alpha helices conformation to mostly beta elements at highest fluence. These changes are accompanied by significant production of carbon monoxide, which was shown to come from heme degradation under irradiation. The radiolytic yields of formation of denatured protein, carbon monoxide, and of heme degradation were determined, and found very close to each other: G+denatured Mb ≈ G+CO ≈ G-heme = 1.6 × 10–8 ± 0.1 × 10–8 mol/J = 0.16 ± 0.01 species/100 eV. The denaturation of the protein to a beta structure and the production of carbon monoxide under ion irradiation are phenomena that may play an important role in the biological effects of ionizing radiation.

Dissimilar effects of the hydrophilic carbon dots on the amyloid aggregation of two model proteins and the mechanism discussion.

Many proteins could aggregate into amyloid fibrils under certain conditions. However, the aggregation process and morphology of the fibrils may be significantly different because of the distinct protein structure. In this article, the hydrophilic carbon dots (Lys-CA-CDs) were prepared using lysine (Lys) and citric acid (CA) as reactant under the assistance of a microwave. The dissimilar modulation effect of Lys-CA-CDs on the aggregation process of distinct structure protein was further investigated, where bovine serum albumin (BSA) and hen egg white lysozyme (HEWL) were chosen as model proteins. All results showed that Lys-CA-CDs displayed the contrary influence on the aggregation process of BSA and HEWL. Lys-CA-CDs could induce BSA to aggregate into more wormlike fibrils and inhibit the aggregation of HEWL into hair-like fibrils. The influence on the aggregation process of BSA may be assigned to the increased concentration of BSA around the Lys-CA-CDs caused by their interaction. However, inserting of Lys-CA-CDs into the inner structure of HEWL led to the change of protein secondary structure. The change of secondary structure further made it difficult for HEWL to aggregate into fibrils and Lys-CA-CDs showed the inhibition effect on HEWL aggregation.

Insighting the effect of lard-based diacylglycerol on the thermal gelation and rheological properties of myofibrillar protein influenced by soy protein isolate

The objective of this study was to investigate the role of the replacing animal fat in the meat gel system with lard-based diacylglycerol (DG), which has health benefits and similar characteristics (such as taste, smell, and usability) to lard triacylglycerol (TG, the major component of pork fat). The impact of lard-based DG on the thermal gelation and rheological properties of myofibrillar protein (MP) as influenced by the incorporation of soy protein isolate (SPI, 0–0.75% protein) was investigated, and the interaction mechanism was also explored. The gel structure became more continuous and compact with the incorporation of DG or purified DG (PDG) and the decrease of SPI content, as reflected by the change of secondary structures, which was beneficial to restrict the mobility of immobilized water. With the incorporation of DG or PDG and the decrease in the amount of SPI, the gel strength, G′, and G″ were increased. Molecular docking results further revealed that the DG interacted with SPI mainly through van der Waals forces and hydrogen bonds, and interacted with MP mainly through van der Waals forces, hydrogen bonds, and hydrophobic interactions. More importantly, the negative binding energies of the DG-protein complexes were lower than those of the TG-protein complexes, indicating that the interaction was spontaneous and that DG is more likely to interact with proteins than TG. In conclusion, this study provides a vital theoretical basis at the molecular level for the development of desired quality meat products containing DG with health benefits.

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Effect of glycosylation on soy protein isolate–sodium carboxymethyl cellulose conjugates heat-induced gels and their applications as carriers of riboflavin

The objective of this study was to improve the rheological properties and riboflavin delivery ability of soy protein isolate (SPI) gels by incorporating sodium carboxymethyl cellulose (CMC) through glycosylation. The investigation focused on the structural and gel properties of SPI–CMC conjugates at different Maillard reaction times (30, 60, 90, 120, and 150 min). The results showed that the degree of grafting initially increased and then decreased as the Maillard reaction times increased, reaching its peak at 90 min (25.52%). The changes in the structure and conformation of the SPI–CMC conjugates were verified by Fourier transform infrared spectroscopy and surface hydrophobicity. An appropriate Maillard reaction reduced the content of highly ordered α-helix and β-sheet while increasing the content of disordered random coil and β-turn. This secondary structural change led to protein unfolding, exposure of surface hydrophobic groups, and orderly aggregation, forming a dense cross-linked gel network. When the Maillard reaction times increased, especially for 90 min, the gel strength and water-holding capacity of the SPI–CMC conjugate gels were significantly increased, along with a denser microstructure. Compared with the SPI and SPI–CMC mixture, SPI–CMC conjugate gels with 60 and 90 min reaction times exhibited notably increased viscosity and storage modulus (G′), as well as better structural recovery characteristics. The above physicochemical property improvements markedly enhanced the riboflavin encapsulation efficiency and riboflavin delivery ability of SPI–CMC conjugate gels, improving the ability to protect riboflavin from photodegradation and slowly releasing riboflavin in the simulated intestinal environment. Hence, SPI–CMC conjugate gels have significant potential for the delivery of nutrients.

Unraveling UVB effects: Catalase activity and molecular alterations in the stratum corneum

AimUltraviolet B (UVB) radiation can compromise the functionality of the skin barrier through various mechanisms. We hypothesize that UVB induce photochemical alterations in the components of the outermost layer of the skin, known as the stratum corneum (SC), and modulate its antioxidative defense mechanisms. Catalase is a well-known antioxidative enzyme found in the SC where it acts to scavenge reactive oxygen species. However, a detailed characterization of acute UVB exposure on the activity of native catalase in the SC is lacking. Moreover, the effects of UVB irradiation on the molecular dynamics and organization of the SC keratin and lipid components remain unclear. Thus, the aim of this work is to characterize consequences of UVB exposure on the structural and antioxidative properties of catalase, as well as on the molecular and global properties of the SC matrix surrounding the enzyme. ExperimentsThe effect of UVB irradiation on the catalase function is investigated by chronoamperometry with a skin covered oxygen electrode, which probes the activity of native catalase in the SC matrix. Circular dichroism is used to explore changes of the catalase secondary structure, and gel electrophoresis is used to detect fragmentation of the enzyme following the UVB exposure. UVB induced alterations of the SC molecular dynamics and structural features of the SC barrier, as well as its water sorption behavior, are investigated by a complementary set of techniques, including natural abundance 13C polarization transfer solid-state NMR, wide-angle X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and dynamic vapor sorption microbalance. FindingsThe findings show that UVB exposure impairs the antioxidative function of catalase by deactivating both native catalase in the SC matrix and lyophilized catalase. However, UVB radiation does not alter the secondary structure of the catalase nor induce any observable enzyme fragmentation, which otherwise could explain deactivation of its function. NMR measurements on SC samples show a subtle increase in the molecular mobility of the terminal segments of the SC lipids, accompanied by a decrease in the mobility of lipid chain trans-gauche conformers after high doses of UVB exposure. At the same time, the NMR data suggest increased rigidity of the polypeptide backbone of the keratin filaments, while the molecular mobility of amino acid residues in random coil domains of keratin remain unaffected by UVB irradiation. The FTIR data show a consistent decrease in absorbance associated with lipid bond vibrations, relative to the main protein bands. Collectively, the NMR and FTIR data suggest a small modification in the composition of fluid and solid phases of the SC lipid and protein components after UVB exposure, unrelated to the hydration capacity of the SC tissue. To conclude, UVB deactivation of catalase is anticipated to elevate oxidative stress of the SC, which, when coupled with subtle changes in the molecular characteristics of the SC, may compromise the overall skin health and elevate the likelihood of developing skin disorders.

KCD: A prediction web server of knowledge-based circular dichroism.

We present a web server that predicts the far-UV circular dichroism (CD) spectra of proteins by utilizing their three-dimensional (3D) structures from the Protein Data Bank (PDB). The main algorithm is based on the classical theory of optical activity together with a set of atomic complex polarizabilities, which are obtained from the analysis of a series of synchrotron radiation CD spectra and their related 3D structures from the PDB. The results of our knowledge-based CD method (KCD) are in good agreement with measured spectra that could include the effect of D-amino acids. Our method also delivers some of the most accurate predictions, in comparison with the calculated spectra from well-established models. Specifically, using a metric of closeness based on normalized absolute deviations between experimental and calculated spectra, the mean values for a series of 57 test proteins give the following figures for such models: 0.26 KCD, 0.27 PDBMD2CD, 0.30 SESCA, and 0.47 DichroCalc. From another point of view, it is worth mentioning the remarkable capabilities of the recent approaches based on artificial intelligence, which can precisely predict the native structure of proteins. The structure of proteins, however, is flexible and can be modified by a diversity of environmental factors such as interactions with other molecules, mechanical stresses, variations of temperature, pH, or ionic strength. Experimental CD spectra together with reliable predictions can be utilized to assess eventual secondary structural changes. A similar kind of evaluation can be done for the case of an incomplete protein structure that has been reconstructed by using different approaches. The KCD method can be freely accessed from: https://kcd.cinvestav.mx/.

Comparative study on interactions of phillyrin and phillygenol with lysozyme: Spectroscopy, differential scanning calorimetry and molecular modeling approaches

Phillyrin (PR) and its derivative phillygenol (PG) are major active extracts of the Chinese medicine Forsythiae Fructus. Lysozyme, an important carrier protein, has a variety of physiological along with pharmaceutical functions. Herein, the interactions of PR and PG with hen egg white lysozyme (HEWL) are investigated in details by spectroscopic analyses, differential scanning calorimetry (DSC) tests, and molecular modeling approaches. Fluorescence measurement and UV–visible absorption difference spectroscopy reveal that an “untypical static quenching” takes place pointing towards the formation of ground state complex. The binding constants of HEWL-PG are greater than that of HEWL-PR. Moreover, the major driving force for the binding process is elucidated as hydrophobic interactions for both cases. Circular dichroism spectroscopy and three-dimensional fluorescence indicate that PR and PG decrease the helical structure of the HEWL and hydrophobicity surrounding Trp and Tyr residues. The thermal stability of HEWL decreases in the presence of PR and PG by DSC. Docking study infers that both PR and PG could bind to the hydrophobic cavity of HEWL and supports the higher binding of PG than PR. Molecular dynamics study illustrates that PR has higher relative position change, which may attribute to its lower binding ability. In terms of the radius of gyration (Rg), PG causes the protein structure to swell even more. The average hydrogen bonds number of HEWL-PG is less than that of HEWL-PR and more changes of the residual fluctuation are observed in HEWL-PG system. Furthermore, MM-PBSA analysis exhibits that HEWL-PG has lower binding energy. Collectively, these results support that both PR and PG could bind to HEWL, but PG possesses higher affinity which leads to stronger secondary structural changes of HEWL than PR. These findings are beneficial to comprehensively understand the binding characteristic of PR and PG with HEWL, providing experimental basis for pharmacokenitics of PR and PG.

Dynamic Study on Water State and Water Migration during Gluten-Starch Model Dough Development under Different Gluten Protein Contents.

Mixing is crucial for dough quality. The gluten content influences water migration in dough development and properties, leading to quality changes in dough-based products. Understanding how the gluten protein content influences water migration during dough development is necessary for dough processing. A compound flour with different gluten protein contents (GPCs, 10-26%, w/w) was used to study the dough farinograph parameters and water migration during dough development. According to the farinograph test of the gluten-starch model dough, the GPC increases the water absorption and the strength of the dough. Water migration was determined via low-field nuclear magnetic resonance (LF-NMR). With the increase in GPC, the gluten protein increases the binding ability of strongly bound water and promotes the transformation of weakly bound water. However, inappropriate GPC (10% and 26%, w/w) results in the release of free water, which is caused by damage to the gluten network according to the microstructure result. Moreover, the changes in proteins' secondary structures are related to the migration of weakly bound water. Therefore, weakly bound water plays an important role in dough development. Overall, these results provide a theoretical basis for the optimization of dough processing.

Abstract 4890: In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals

Abstract Some mammals such as elephants, bats, whales, and blind mole rats (Spalax) are resistant to cancer. One of the cancer protective mechanisms present in such species includes a reduced degradation of heparan sulfate (HS), an important component of the extracellular matrix (ECM), present in Spalax. Such effect occurs due to a splicing variant of heparanase, called variant 36, that lacks enzymatic activity. Consequently, both HS and ECM are very stable in Spalax’s tissues. If HS is stabilized, in cancer resistant mammals, due to the decrease of heparanase or other enzymes involved in HS catabolic process is unknown. We studied total of 9 enzymes (HPSE2, GusB, IDUA, IDS, NAGLU, SGSH, GNS, Sulf-2, HGSNAT) that regulate HS catabolism and homologs in 9 mammals. Multiple sequence alignments were done by MEGA to identify substitutions present in cancer resistant species in comparison with cancer susceptible animals. Such amino acid variants were further analyzed and both their energy stability and their functional effects were predicted by using online in silico tools including MAESTROweb, Condel, PANTHER, REVEL, and CADD. Finally, homologs with deleterious predicted variants were modelled with SWISSMODEL and their 3D structures were analyzed with Pymol and ChimeraX to identify structural features likely related to its loss of function. We found two loss of function missense variants, K264Q and W626L, in Alpha-L-iduronidase (IDUA) of Spalax. In addition, most of Brandt's bat enzymes and the N-sulphoglucosamine sulphohydrolase (SGSH) of the bowhead whale were very energy destabilizing enzymes with positive and higher ΔΔG values compared to human homologs. In the other hand, although some catalytic residues in the study enzymes are conserved in cancer resistant animals, their spatial arrangement and conformation in the 3D structure are different compared to human homologs, including E182 of IDUA which is one of the catalytic residues. In general, the secondary structure is conserved across the 9 enzymes studied but some differences were observed in cancer resistant animals including one of the beta strands near the catalytic domain in human and mice IDUA that appears as a loop in Spalax. In summary, HS stabilization could raise in cancer resistant mammals through the existence of lack of enzymatic activity not only in heparanase but also in other enzymes that participate in HS catabolic process, via a set of loss of function variants and changes in secondary and tertiary structure as well as a reduction of energy stabilization. More interestingly, such mechanism could be present in other cancer resistant mammals such as bats and bowhead whales. This study demonstrates that computational approaches are useful to study biological mechanisms of cancer resistance present in some mammals. This knowledge could be translated to humans and HS catabolic process could offer a new source of therapeutic anticancer targets. Citation Format: Fabrizio Oliveri Payet, Claudia Machicado Rivero. In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4890.

5-methyl-salicylaldoxime based ionic liquids for non-destructive separation of protein and cadmium

Confronting with explosive cadmium contaminations to environment, non-destructive separation of protein with cadmium for protecting food safety is hardpressed. Ionic liquids (ILs) are benign bio-compatible stabilizer of proteins with considerable metal affinity, while their application in separation of cadmium from protein is limited owing to their comparative metal affinities with protein. Accordingly, we firstly synthesize 1-methyl-3 (salicylaldoxime-5-methyl) imidazole hexafluorophosphate ([Salenmim][PF6]) to enhance cadmium extraction with hydroxyl and oxime groups as ligands of cadmium. Structure of [Salenmim][PF6] is confirmed by 1H NMR, 13C NMR, 31P NMR, 19F NMR, FT-IR, Raman spectra, and theoretical simulations. [Salenmim][PF6] can remove 85.31 % of Cd2+ from bovine serum albumin (BSA) at pH of 2.0. X-ray photoelectron spectroscopy (XPS) of BSA and [Salenmim][PF6] before and after Cd2+ extraction illustrate that Cd2+ generally bonds with C = N and O-H of [Salenmim][PF6] to achieve detachment of Cd2+ from BSA. Molecular docking of BSA and Cd2+ indicates that 4 amino acids residues listed as ASP561, ASP562, LYS563, and GLU564 coordinate with Cd2+ with coordinate number close to 1, further confirmed as 1.48 by isothermal titration calorimetry (ITC) analysis. DFT calculations of [Salenmim][PF6] with Cd2+ demonstrate that energy gap between HOMO orbital localized at benzene ring is 1.6860 eV higher than LUMO orbital, faciliating formation of [Salenmim][PF6]-Cd2+ complex. Finally, there are no changes in secondary and tertiary structure of BSA and no ILs residual observed after [Salenmim][PF6] assisted Cd2+ extraction. This work firstly reports a method for synthesis of [Salenmim][PF6] with high affinity to cadmium and systemically reveals how do structure of [Salenmim][PF6] enhance heavy metal removal from proteins, providing a possible strategy for heavy metal extraction from proteins.