HLA-B and TIMP1 as hub genes of the ventricular remodeling caused by hypertension.

RATIONALE: Myocardial fibrosis is an important pathological change that occurs during ventricular remodeling in patients with hypertension and is an important pathophysiological basis of cardiovascular disease. However, the molecular mechanism underlying this ventricular remodeling is unclear. METHODS: Bioinformatics analysis identified HLA-B and TIMP1 as hub genes in the process of myocardial fibrosis. Expression and correlation analyses of significant hub genes with ventricular remodeling were performed. Weighted gene co-expression network analysis (WGCNA) was performed to verify the role of HLA-B. ceRNA network was constructed to identify the candidate molecule drugs. Receiver operating characteristic (ROC) curves were analyzed. RESULTS: RT-qPCR was performed to verify the roles of HLA-B and TIMP1 in seven control individuals with hypertension and seven patients with hypertension and ventricular remodeling. The WGCNA showed that HLA-B was in the brown module and the correlation coefficient between HLA-B and ventricular remodeling was 0.67. Based on univariate logistic proportional regression analysis, HLA-B influences ventricular remodeling (P<0.05). RT-qPCR showed that the relative expression levels of HLA-B and TIMP1 were significantly higher in HLVR samples compared with their expression in the control group. CONCLUSIONS: HLA-B and TIMP1 might provide novel research targets for the diagnosis and treatment of HLVR.

Higher expression of TSR2 aggravating hypertension via the PPAR signaling pathway.

Hypertension is a complex disease with unknown causes. Therefore, it's crucial to deeply study its molecular mechanism. The hypertension dataset was obtained from Gene Expression Omnibus data base (GEO), and miRNA regulating central hub genes was screened via weighted gene co-expression network (DEGs) and gene set enrichment (GSEA). Cell experiments validated TSR2's role and the PPAR signaling pathway through western blotting. 500 DEGs were identified for hypertension, mainly enriched in actin cross-linking, insulin signaling, PPAR signaling, and protein localization. Eight hub genes (SEC61G, SRP14, Liy AR, NIP7, SDAD1, POLR1D, DYNLL2, TSR2) were identified. Four hub genes (LYAR, SDAD1, POLR1D, TSR2) exhibited high expression levels in the hypertensive tissue samples, while showing low expression levels in the normal tissue samples. This led us to speculate that they may have relevant regulatory effects on hypertension. When TSR2 was knocked down in the hypertension peripheral blood mononuclear cells (PBMC) model, the critical proteins in the PPAR signaling pathway (FABP, PPAR, PLTP, ME1, SCD1, CYP27, FABP1, OLR1, CPT-1, PGAR, CAP, ADIPO, MMP1, UCP1, ILK, PDK1 UBC AQP7) were downregulated. This also occurred in the hypertension peripheral blood mononuclear cells (PBMC) + TSR2_ OV model. TSR2 is highly expressed in individuals with hypertension and may play a significant role in the development of hypertension through the PPAR signaling pathway. TSR2 could serve as a molecular target for the early diagnosis and precise treatment of hypertension, providing a valuable direction for the mechanism research of this condition.

Unnatural Amino Acids for Biological Spectroscopy and Microscopy.

Due to advances in methods for site-specific incorporation of unnatural amino acids (UAAs) into proteins, a large number of UAAs with tailored chemical and/or physical properties have been developed and used in a wide array of biological applications. In particular, UAAs with specific spectroscopic characteristics can be used as external reporters to produce additional signals, hence increasing the information content obtainable in protein spectroscopic and/or imaging measurements. In this Review, we summarize the progress in the past two decades in the development of such UAAs and their applications in biological spectroscopy and microscopy, with a focus on UAAs that can be used as site-specific vibrational, fluorescence, electron paramagnetic resonance (EPR), or nuclear magnetic resonance (NMR) probes. Wherever applicable, we also discuss future directions.

Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences.

Triple bonds, such as that formed between two carbon atoms (i.e., C≡C) or that formed between one carbon atom and one nitrogen atom (i.e., C≡N), afford unique chemical bonding and hence vibrational characteristics. As such, they are not only frequently used to construct molecules with tailored chemical and/or physical properties but also employed as vibrational probes to provide site-specific chemical and/or physical information at the molecular level. Herein, we offer our perspective on the emerging applications of various triple-bond vibrations in energy and biological sciences with a focus on C≡C and C≡N triple bonds.

C≡N Stretching Frequency as a Convenient Reporter of Charge Separation in Molecular Systems.

Previously, several studies have shown that, for a set of structurally related nitrile compounds, there could be a linear relationship between the total charge on the nitrile group (qCN) and its stretching frequency (νCN). However, it is unclear whether the corresponding frequency and charge properties of structurally different nitrile compounds can be described by a single linear νCN-qCN relationship. Herein, we compute the qCN magnitudes of a large number of nitrile-containing molecules whose νCN values cover a spectral range of ca. 200 cm-1 and are measured under different experimental conditions. Our results reveal that νCN indeed exhibits a linear dependence on qCN, with a slope of 637 ± 30 cm-1/charge. Because the nitrile moiety is a commonly used building block in electronic donor-acceptor (D-A) molecular systems, we believe that this linear relationship will find utility in a wide range of applications where such D-A constructs are used, such as in organic photovoltaic assemblies. In addition, we apply this linear relationship to characterize the degree of charge transfer upon photoexcitation of two indole derivatives, 5-cyanoindole and 6-cyanoindole, and are able to show that in both cases, the fluorescence emission arises from a charge-transfer or La state.

Photophysics of Two Indole-Based Cyan Fluorophores.

For the purpose of searching for new biological fluorophore, we assess the photophysical properties of two indole derivatives, 4-cyano-7-azaindole (4CN7AI) and 1-methyl-4-cyano-7-azaindole (1M4CN7AI), in a series of solvents. We find that (1) the absorption spectra of both derivatives are insensitive to solvents and are red-shifted from that of indole, having a maximum absorption wavelength of ca. 318 nm and a broad profile that extends beyond 370 nm; (2) both derivatives emit in the blue to green spectral range with a large Stokes shift, for example, in H2O, the maximum emission wavelength of 4CN7AI (1M4CN7AI) is at ca. 455 nm (470 nm); (3) 4CN7AI has a higher fluorescence quantum yield (QY) and a longer fluorescence lifetime (τF) in aprotic solvents than in protic solvents, for example, QY (τF) = 0.72 ± 0.04 (7.6 ± 0.8 ns) in tetrahydrofuran and QY (τF) = 0.29 ± 0.03 (6.2 ± 0.6 ns) in H2O; (4) in all of the solvents used except H2O, the fluorescence QY (τF) of 1M4CN7AI is equal to or higher (longer) than 0.69 ± 0.03 (11.2 ± 0.7 ns). Taken together, these results suggest that the corresponding non-natural amino acids, 4-cyano-7-azatryptophan and 1-methyl-4-cyano-7-azatryptophan, could be useful as biological fluorophores.

4-Cyanotryptophan as a Sensitive Fluorescence Probe of Local Electric Field of Proteins.

Electrostatic interactions are key determinants of protein structure, dynamics, and function. Since protein electrostatics are nonuniform, assessment of the internal electric fields (EFs) of proteins requires spatial resolution at the amino acid residue level. In this regard, vibrational Stark spectroscopy, in conjunction with various unnatural amino acid-based vibrational probes, has become a common method for site-specific interrogation of protein EFs. However, application of this method is often limited to proteins with relatively high solubility, due to the intrinsically low oscillator strength of vibrational transitions. Therefore, it would be useful to develop an alternative method that can overcome this limitation. To this end, we show that, using solvatochromic study and molecular dynamics simulations, the frequency of maximum emission intensity of the fluorophore of 4-cyanotryptophan (4CN-Trp), 3-methyl-1H-indole-4-carbonitrile, exhibits a linear dependence on the local EF. Since the absorption and emission spectra of 4CN-Trp are easily distinguishable from those of naturally occurring aromatic amino acids, we believe that this linear relationship provides an easier and more sensitive means to determine the local EF of proteins.

Photoenhancement of the C≡N Stretching Vibration Intensity of Aromatic Nitriles.

The C≡N stretching vibration is a versatile infrared (IR) reporter that is useful for a wide range of applications. Aiming to further expand its spectroscopic utility, herein, we show that, using 4-cyanoindole and 4-cyano-7-azaindole as examples, photoexcitation can significantly shift the frequency (νCN) and enhance the molar extinction coefficient (εCN) of this vibrational mode of aromatic nitriles and that, for these indole derivatives, the enhancement factor can reach 13. Moreover, we find that while solvent relaxation at the excited electronic state(s) always leads to an increase in εCN, its effect on νCN depends on the solute and the solvent. Taken together, these results demonstrate that solvent relaxation can differently affect the local environment of the nitrile group and its conjugation with the indole ring and, more importantly, that the C≡N stretching vibration can serve as a sensitive IR probe of charge and electron transfer processes in which an aromatic nitrile is involved.

SGLT1/2 as the potential biomarkers of renal damage under Apoe-/- and chronic stress via the BP neural network model and support vector machine.

Background: Chronic stress (CS) could produce negative emotions. The molecular mechanism of SGLT1 and SGLT2 in kidney injury caused by chronic stress combined with atherosclerosis remains unclear. Methods: In total, 60 C57BL/6J mice were randomly divided into four groups, namely, control (CON, n = 15), control diet + chronic stress (CON+CS, n = 15), high-fat diet + Apoe-/- (HF + Apoe-/-, n = 15), and high-fat diet + Apoe-/- + chronic stress (HF+Apoe-/- + CS, n = 15) groups. The elevated plus maze and open field tests were performed to examine the effect of chronic stress. The expression of SGLT1 and SGLT2 in the kidney was detected. The support vector machine (SVM) and back propagation (BP) neural network model were constructed to explore the predictive value of the expression of SGLT1/2 on the renal pathological changes. The receiver operating characteristic (ROC) curve analysis was used. Results: A chronic stress model and atherosclerosis model were constructed successfully. Edema, broken reticular fiber, and increased glycogen in the kidney would be obvious in the HF + Apoe-/- + CS group. Compared with the CON group, the expression of SGLT1/2 in the kidney was upregulated in the HF + Apoe-/- + CS group (P < 0.05). There existed positive correlations among edema, glycogen, reticular fiber, expression of SGLT1/2 in the kidney. There were higher sensitivity and specificity of diagnosis of SGLT1/2 for edema, reticular fiber, and glycogen in the kidney. The result of the SVM and BP neural network model showed better predictive values of SGLT1 and SGLT2 for edema and glycogen in the kidney. Conclusion: In conclusion, SGLT1/2 might be potential biomarkers of renal damage under Apoe-/- and chronic stress, which provided a potential research direction for future related explorations into this mechanism.

A Potential Target for Clinical Atherosclerosis: A Novel Insight Derived from TPM2.

Atherosclerosis (AS) is a potential inducer of numerous cardio-cerebrovascular diseases. However, little research has investigated the expression of TPM2 in human atherosclerosis samples. A total of 34 clinical samples were obtained, including 17 atherosclerosis and 17 normal artery samples, between January 2018 and April 2021. Bioinformatics analysis was applied to explore the potential role of TPM2 in atherosclerosis. Immunohistochemistry, immunofluorescence, and western blotting assays were used to detect the expression of TPM2 and α-SMA proteins. The mRNA expression levels of TPM2 and α-SMA were detected using RT-qPCR. A neural network and intima-media thickness model were constructed. A strong relationship existed between the intima-media thickness and relative protein expression of TPM2 (P<0.001, R=-0.579). The expression of TPM2 was lower in atherosclerosis than normal artery (P<0.05). Univariate logistic regression showed that TPM2 (OR=0.150, 95% CI: 0.026-0.868, P=0.034) had clear correlations with atherosclerosis. A neural network model was successfully constructed with a relativity of 0.94434. TPM2 might be an independent protective factor for arteries, and one novel biomarker of atherosclerosis.

Site-Specific Interrogation of Protein Structure and Stability.

To execute their function or activity, proteins need to possess variability in local electrostatic environment, solvent accessibility, structure, and stability. However, assessing any protein property in a site-specific manner is not easy since native spectroscopic signals often lack the needed specificity. One strategy that overcomes this limitation is to use unnatural amino acids that exhibit distinct spectroscopic features. In this chapter, we describe several such unnatural amino acids (UAAs) and their respective applications in site-specific interrogation of protein structure and stability using standard biophysical methods, including circular dichroism (CD), infrared (IR), and fluorescence spectroscopies.

Citrate Synthase and OGDH as Potential Biomarkers of Atherosclerosis under Chronic Stress.

BACKGROUND: Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). METHODS: New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of genes. In addition, a neural network model was constructed. The quantitative relationships were inferred by cubic spline interpolation. Enzymatic activity of mitochondrial citrate synthase and OGDH was detected by the enzymatic assay kit. Function of citrate synthase and OGDH with knockdown experiments in the adrenal cell lines was performed. Furthermore, target genes-TF-miRNA regulatory network was constructed. Coimmunoprecipitation (IP) assay and molecular docking study were used to detect the interaction between citrate synthase and OGDH. RESULTS: Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH was higher in the AS+CS group than in the control group, both at the protein and mRNA levels (P < 0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P < 0.05) via Spearman's rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Enzymatic activity of citrate synthase and OGDH increased under the situation of atherosclerosis and chronic stress. Through the CCK8 assay, the adrenal cell viability was downregulated significantly after the knockdown experiment of citrate synthase and OGDH. Target genes-TF-miRNA regulatory network presented the close interrelations among the predicted microRNA, citrate synthase and OGDH. After Coimmunoprecipitation (IP) assay, the result manifested that the citrate synthase and OGDH were coexpressed in the adrenal gland. The molecular docking study showed that the docking score of optimal complex conformation between citrate synthase and OGDH was -6.15 kcal/mol. CONCLUSION: AS combined with CS plays a significant role on the hypothalamic-pituitary-adrenal (HPA) axis, promotes adrenomegaly, increases the release of glucocorticoid (GC), and might enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future related explorations into this mechanism.

Tryptophan as a Template for Development of Visible Fluorescent Amino Acids.

Most biological systems, at both molecular and cellular levels, are intrinsically complex, diverse, and nonfluorescent. Therefore, studying their structures, dynamics, and interactions via fluorescence-based methods requires incorporation of one or multiple external fluorophores that would not significantly affect any native property of the system in question. This requirement necessitates the development of a diverse set of fluorescence reporters that differ in chemical, physical, and photophysical properties. Herein, we offer our perspective on the need for, recent progress in, and future directions of developing tryptophan-based fluorescent amino acids.

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid.

The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

Tuning the electronic transition energy of indole via substitution: application to identify tryptophan-based chromophores that absorb and emit visible light.

Fluorescent amino acids (FAAs) offer significant advantages over fluorescent proteins in applications where the fluorophore size needs to be limited or minimized. A long-sought goal in biological spectroscopy/microcopy is to develop visible FAAs by modifying the indole ring of tryptophan. Herein, we examine the absorption spectra of a library of 4-substituted indoles and find that the frequency of the absorption maximum correlates linearly with the global electrophilicity index of the substituent. This finding permits us to identify two promising candidates, 4-formyltryptophan (4CHO-Trp) and 4-nitrotryptophan (4NO2-Trp), both of which can be excited by visible light. Further fluorescence measurements indicate that while 4CHO-indole (and 4CHO-Trp) emits cyan fluorescence with a reasonably large quantum yield (ca. 0.22 in ethanol), 4NO2-indole is essentially non-fluorescent, suggesting that 4CHO-Trp (4NO2-Trp) could be useful as a fluorescence reporter (quencher). In addition, we present a simple method for synthesizing 4CHO-Trp.

A Scalable Synthesis of the Blue Fluorescent Amino Acid 4-Cyanotryptophan and the Fmoc Derivative: Utility Demonstrated with the Influenza M2 Peptide Tetramer.

A scalable synthesis of the Fmoc-protected blue fluorescent amino acid, l-4-cyanotryptophan (W4CN), that exploits an enantioselective phase transfer-catalyzed alkylation is reported. The red-shifted emission of water-exposed W4CN residues was leveraged to investigate the solvation state of tryptophan (Trp) residues within the influenza M2 proton channel. The correlation of the channel's conformation (i.e., open or closed) with the fluorescence spectrum of a mutated W4CN residue suggests that the channel's conformational state does not impact the hydration status of the Trp residues.

Assessing the Effect of Hofmeister Anions on the Hydrogen-Bonding Strength of Water via Nitrile Stretching Frequency Shift.

The temperature dependence of the peak frequency (νmax) of the C≡N stretching vibrational spectrum of a hydrogen-bonded C≡N species is known to be a qualitative measure of its hydrogen-bonding strength. Herein, we show that within a two-state framework, this dependence can be analyzed in a more quantitative manner to yield the enthalpy and entropy changes (ΔHHB and ΔSHB) for the corresponding hydrogen-bonding interactions. Using this method, we examine the effect of ten common anions on the strength of the hydrogen-bond(s) formed between water and the C≡N group of an unnatural amino acid, p-cyanophenylalanine (PheCN). We find that based on the ΔHHB values, these anions can be arranged in the following order: HPO42- > OAc- > F- > SO42- ≈ Cl- ≈ (H2O) ≈ ClO4- ≈ NO3- > Br- > SCN- ≈ I-, which differs from the corresponding Hofmeister series. Because PheCN has a relatively small size, the finding that anions having very different charge densities (e.g., SO42- and ClO4-) act similarly suggests that this ranking order is likely the result of specific ion effects. Since proteins contain different backbone and side-chain units, our results highlight the need to assess their individual contributions toward the overall Hofmeister effect in order to achieve a microscopic understanding of how ions affect the physical and chemical properties of such macromolecules. In addition, the analytical method described in the present study is applicable for analyzing the spectral evolution of any vibrational spectra composed of two highly overlapping bands.

4-Cyanoindole-based fluorophores for biological spectroscopy and microscopy.

Most biological molecules are intrinsically non- or weakly-fluorescent, hence requiring labeling with an external fluorophore(s) to be studied via fluorescence-based techniques. However, such labeling could perturb the native property of the system in question. One effective strategy to minimize such undesirable perturbation is to use fluorophores that are simple analogs of natural amino acids. In this chapter, we describe the synthesis and spectroscopic utility of two indole-based fluorophores, 4-cynaotryprophan (4CN-Trp) and 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), with a focus on 4CN-Trp. This unnatural amino acid, which is only slightly larger than its natural counterpart, tryptophan (Trp), exhibits unique photophysical properties, making it a versatile fluorophore in biological spectroscopic and imaging applications. Through several specific examples, we highlight its broad utility in the study of various biological problems and processes.

Can glycine betaine denature proteins?

Glycine betaine (GB) is a naturally occurring osmolyte that has been widely recognized as a protein protectant. Since GB consists of a methylated ammonium moiety, it can engage in strong cation-π interactions with aromatic amino acid sidechains. We hypothesize that such specific binding interactions would allow GB to decrease the stability of proteins that are predominantly stabilized by a cluster of aromatic amino acids. To test this hypothesis, we investigate the effect of GB on the stability of two ß-hairpins (or mini-proteins) that contain such a cluster. We find that for both systems the stability of the folded state first decreases and then increases with increasing GB concentration. Such non-monotonic dependence not only confirms that GB can act as a protein denaturant, but also underscores the complex interplay between GB's stabilizing and destabilizing forces toward a given protein. While stabilizing osmolytes all have the tendency to be excluded from the protein surface which is the action underlying their stabilizing effect, our results suggest that in order to quantitatively assess the effect of GB on the stability of any given protein, specific cation-π binding interactions need to be explicitly considered. Moreover, our results show, consistent with other studies, that cation methylation can strengthen the respective cation-π interactions. Taken together, these findings provide new insight into the mechanism by which amino acid-based osmolytes interact with proteins.

PET and FRET utility of an amino acid pair: tryptophan and 4-cyanotryptophan.

Methods based on fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) are widely used in the biological sciences, employing mostly dye-based FRET and PET pairs. While very useful and important, dye-based reporters are not always applicable without concern, for example, in cases where the fluorophore size needs to be minimized. Therefore, development and characterization of smaller, ideally amino acid-based PET and FRET pairs will expand the biological spectroscopy toolbox to enable new applications. Herein, we show that, depending on the excitation wavelength, tryptophan and 4-cyanotrptophan can interact with each other via the mechanism of either energy or electron transfer, hence constituting a dual FRET and PET pair. The biological utility of this amino acid pair is further demonstrated by applying it to study the end-to-end collision rate of a short peptide, the mode of interaction between a ligand and BSA, and the activity of a protease.