Amino Acids (v.50, #5)

Ameliorative effects of taurine against diabetes: a review by Inam-u-llah; Fengyuan Piao; Rana Muhammad Aadil; Raheel Suleman; Kaixin Li; Mengren Zhang; Pingan Wu; Muhammad Shahbaz; Zulfiqar Ahmed (487-502).
Diets in rats and humans have shown promising results. Taurine improved glucagon activity, promoted glycemic stability, modified glucose levels, successfully addressed hyperglycemia via advanced glycation end-product control, improved insulin secretion and had a beneficial effect on insulin resistance. Taurine treatment performed well against oxidative stress in brain, increased the secretion of required hormones and protected against neuropathy, retinopathy and nephropathy in diabetes compared with the control. Taurine has been observed to be effective in treatments against diabetic hepatotoxicity, vascular problems and heart injury in diabetes. Taurine was shown to be effective against oxidative stress. The mechanism of action of taurine cannot be explained by one pathway, as it has many effects. Several of the pathways are the advanced glycation end-product pathway, PI3-kinase/AKT pathway and mitochondrial apoptosis pathway. The worldwide threat of diabetes underscores the urgent need for novel therapeutic measures against this disorder. Taurine (2-aminoethane sulfonic acid) is a natural compound that has been studied in diabetes and diabetes-induced complications.
Keywords: Taurine; Diabetes; Neuropathy; Nephropathy; Retinopathy

Evaluation of peptides release using a natural rubber latex biomembrane as a carrier by M. C. R. Miranda; F. A. Borges; N. R. Barros; N. A. Santos Filho; R. J. Mendonça; R. D. Herculano; E. M. Cilli (503-511).
The biomembrane natural (NRL—Natural Rubber Latex), manipulated from the latex obtained from the rubber tree Hevea brasiliensis, has shown great potential for application in biomedicine and biomaterials. Reflecting the biocompatibility and low bounce rate of this material, NRL has been used as a physical barrier to infectious agents and for the controlled release of drugs and extracts. The aim of the present study was to evaluate the incorporation and release of peptides using a latex biomembrane carrier. After incorporation, the release of material from the membrane was observed using spectrophotometry. Analyses using HPLC and mass spectroscopy did not confirm the release of the antimicrobial peptide [W6]Hylin a1 after 24 h. In addition, analysis of the release solution showed new compounds, indicating the degradation of the peptide by enzymes contained in the latex. Additionally, the release of a peptide with a shorter sequence (Ac-WAAAA) was evaluated, and degradation was not observed. These results showed that the use of NRL as solid matrices as delivery systems of peptide are sequence dependent and could to be evaluated for each sequence.
Keywords: Drug delivery; Peptides; Natural rubber latex; Biomaterial

Hepatic glutamate uptake and conversion to glutamine is critical for whole-body N metabolism, but how this process is regulated during growth is poorly described. The hepatic glutamate uptake activities, protein content of system $${ ext{X}}^{ - }_{ ext{AG}}$$ XAG- transporters (EAAC1, GLT-1) and regulatory proteins (GTRAP3-18, ARL6IP1), glutamine synthetase (GS) activity and content, and glutathione (GSH) content, were compared in liver tissue of weaned Angus steers randomly assigned (n = 8) to predominantly lean (growing) or predominantly lipid (finished) growth regimens. Steers were fed a cotton seed hull-based diet to achieve final body weights of 301 or 576 kg, respectively, at a constant rate of growth. Liver tissue was collected at slaughter and hepatic membranes fractionated. Total (75%), Na+-dependent (90%), system $${ ext{X}}^{ - }_{ ext{AG}}$$ XAG- -dependent (abolished) glutamate uptake activity, and EAAC1 content (36%) in canalicular membrane-enriched vesicles decreased as steers developed from growing (n = 6) to finished (n = 4) stages, whereas Na+-independent uptake did not change. In basolateral membrane-enriched vesicles, total (60%), Na+-dependent (60%), and Na+-independent (56%) activities decreased, whereas neither system $${ ext{X}}^{ - }_{ ext{AG}}$$ XAG- -dependent uptake nor protein content changed. EAAC1 protein content in liver homogenates (n = 8) decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased (32%) as steers fattened, whereas GS and GSH contents did not differ. We conclude that hepatic glutamate uptake and GS synthesis capacities are reduced in livers of finished versus growing beef steers, and that hepatic system $${ ext{X}}^{ - }_{ ext{AG}}$$ XAG- transporter activity/EAAC1 content is inversely proportional to GTRAP3-18 content.
Keywords: Cattle; Glutamate transport; Glutamine synthetase; Growth phase; Liver

Taurine is an amino acid with the ability to activate autophagy in adipocytes by Hiroki Kaneko; Masaki Kobayashi; Yuhei Mizunoe; Maho Yoshida; Hiromine Yasukawa; Shunsuke Hoshino; Rei Itagawa; Takuma Furuichi; Naoyuki Okita; Yuka Sudo; Masato Imae; Yoshikazu Higami (527-535).
Alterations in adipocyte characteristics are highly implicated in the pathology of obesity. In a recent article, we demonstrated that high-fat diet-induced obesity impairs lysosomal function, thereby suppressing autophagy in mice white adipose tissue. Taurine, an amino acid naturally contained in the normal diet and existing ubiquitously in tissues, has been reported to improve insulin resistance and chronic inflammation in animal models, but underlying mechanisms remain unclear. From these findings, we hypothesized that improvement of obese pathology by taurine may be mediated through recovery of autophagy. In matured 3T3-L1 mouse adipocytes, treatment with taurine-promoted autophagy. Moreover, taurine-induced nuclear translocation of transcription factor EB (TFEB), a master regulator of autophagy- and lysosome-related factors. As this translocation is regulated by several kinase pathways, including extracellular signal-related kinase 1 and 2 (ERK1/2) and mechanistic target of rapamycin protein kinase complex 1 (MTORC1), we examined related signaling elements. Consequently, taurine-reduced phosphorylation levels of ERK1/2 but did not alter the phosphorylation of MTORC1 pathway-associated adenosine monophosphate-activated protein kinase or ribosomal protein S6 kinase. Taken together, these results suggest that taurine may enhance TFEB nuclear translocation through ERK1/2 to accelerate autophagy. The effect discovered in this study may represent a novel mechanism for the improvement of obesity-related pathology by taurine.
Keywords: Taurine; Obesity; Adipocyte; Autophagy; TFEB

Demethylation of methionine and keratin damage in human hair by Kamila Borowczyk; Joanna Suliburska; Hieronim Jakubowski (537-546).
Growing human head hair contains a history of keratin and provides a unique model for studies of protein damage. Here, we examined mechanism of homocysteine (Hcy) accumulation and keratin damage in human hair. We found that the content of Hcy-keratin increased along the hair fiber, with levels 5–10-fold higher levels in older sections at the hair’s tip than in younger sections at hair’s base. The accumulation of Hcy led to a complete loss of keratin solubility in sodium dodecyl sulfate. The increase in Hcy-keratin was accompanied by a decrease in methionine-keratin. Levels of Hcy-keratin were correlated with hair copper and iron in older hair. These relationships were recapitulated in model experiments in vitro, in which Hcy generation from Met exhibited a similar dependence on copper or iron. Taken together, these findings suggest that Hcy-keratin accumulation is due to copper/iron-catalyzed demethylation of methionine residues and contributes to keratin damage in human hair.
Keywords: Hair keratin damage; Homocysteine; Methionine demethylation; Copper; Iron

Beyond nigrostriatal dopaminergic system, the noradrenergic locus coeruleus (LC/NE) neurons are also degenerated in patients with Parkinson’s disease (PD), the second most common neurodegenerative disorder. We previously reported that microglia-mediated neuroinflammation contributes to LC/NE neurodegeneration. The purpose of this study is aimed to test whether taurine, an endogenous amino acid, could be able to protect LC/NE neurons through inhibition of microglial activation using paraquat and maneb-induced mouse PD model. Taurine (150 mg/kg) was administrated (i.p) to mice 30 min prior to paraquat (10 mg/kg) and maneb (30 mg/kg) intoxication for consecutive 6 weeks (twice per week). The results clearly demonstrated that paraquat and maneb co-exposure resulted in loss of tyrosine hydroxylase-positive neurons in the LC in mice, which was significantly ameliorated by taurine. Mechanistically, inhibition of microglia-mediated neuroinflammation contributed to taurine-afforded neuroprotection. Taurine attenuated paraquat and maneb-induced microglial activation and M1 polarization as well as release of proinflammatory cytokines in brainstem of mice. Taurine also abrogated microglial NADPH oxidase activation and oxidative damage in paraquat and maneb-treated mice. Furthermore, inhibition of nuclear factor-κB (NF-κB) but not signal transducers and activators of transcription 1/3 (STAT1/3) signaling pathway participated in taurine-inhibited microglial activation. Collectively, taurine exerted LC/NE neuroprotection against microglia-mediated neurotoxicity. The robust neuroprotective effects of taurine suggest that taurine may be a promising candidate for potential therapy for patients suffering from PD.
Keywords: Locus coeruleus; Noradrenergic neuron; Parkinson’s disease; Neuroinflammation; NADPH oxidase

Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan by P. Carvajal-Rondanelli; M. Aróstica; C. A. Álvarez; C. Ojeda; F. Albericio; L. F. Aguilar; S. H. Marshall; F. Guzmán (557-568).
Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.
Keywords: Lysine homopeptide-antimicrobial activity-Ala; Pro scanning-membrane rupture

Comparative effects of acute-methionine loading on the plasma sulfur-amino acids in NAC-supplemented HIV+ patients and healthy controls by Roberto Carlos Burini; Maria Doroteia Borges-Santos; Fernando Moreto; Yong- Ming Yu (569-576).
In this study, an acute overloading of methionine (MetLo) was used to investigate the trassulfuration pathway response comparing healthy controls and HIV+ patients under their usual diet and dietary N-acetyl-l-cysteine (NAC) supplementation. MetLo (0.1 g Met/kg mass weight) was given after overnight fasting to 20 non-HIV+ control subjects (Co) and 12 HIV+ HAART-treated patients. Blood samples were taken before and after the MetLo in two different 7-day dietary situations, with NAC (1 g/day) or with their usual diet (UD). The amino acids (Met, Hcy, Cys, Tau, Ser, Glu and Gln) and GSH were determined by HPLC and their inflow rate into circulation (plasma) was estimated by the area under the curve (AUC). Under UD, the HIV+ had lower plasma GSH and amino acids (excepting Hcy) and higher oxidative stress (GSSG/GSH ratio), similar remethylation (RM: Me/Hcy + Ser ratio), transmethylation (TM; Hcy/Met ratio) and glutaminogenesis (Glu/Gln ratio), lower transsulfuration (TS: Cys/Hcy + Ser ratio) and Cys/Met ratio and, higher synthetic rates of glutathione (GG: GSH/Cys ratio) and Tau (TG: Tau/Cys ratio). NAC supplementation changed the HIV pattern by increasing RM above control, normalizing plasma Met and TS and, increasing plasma GSH and GG above controls. However, plasma Cys was kept always below controls probably, associatively to its higher consumption in GG (more GSSG than GSH) and TG. The failure of restoring normal Cys by MetLo, in addition to NAC, in HIV+ patients seems to be related to increased flux of Cys into GSH and Tau pathways, probably strengthening the cell-antioxidant capacity against the HIV progression (registered at, NCT00910442).
Keywords: HIV+ patients; Methionine-load test; Thiol redox system; Cysteine metabolism

Selection and identification of novel peptides specifically targeting human cervical cancer by Xiaomin Liu; Jingwen Peng; Jie He; Qiaoran Li; Jianbin Zhou; Xiaoqiu Liang; Shengsong Tang (577-592).
Cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer deaths among females in underdeveloped countries. This study aimed to identify several novel cervical cancer-specific targeting peptides (CSPs) to provide new methods for the effective diagnosis and treatment of cervical cancer. Peptide library screening in vivo was performed on human cervical cancer xenografts with Ph.D.™-12 and C7C phage display peptide libraries. Two specific peptide sequences (GDALFSVPLEVY and KQNLAEG), which were enriched in tumors, were screened, and respectively, named CSP-GD and CSP-KQ through three rounds of biopanning. The in vivo tumor-targeting ability of these peptides was identified by injecting them into mice with cervical cancer xenograft. CSPs were compounded and labeled with fluorescein isothiocyanate (FITC). The specificity and affinity of FITC-CSPs were evaluated in human cervical cancer cell lines and tissue microarrays in vitro by immunofluorescent staining. Results showed that FITC-CSP-GD and FITC-CSP-KQ evidently and specifically bound to the cell membrane and cytoplasm of SiHa, ME-180, and C-33A cells in vitro. In human cervical cancer tissue, FITC-CSP-GD and FITC-CSP-KQ strongly targeted human cervical adenocarcinoma and cervical squamous cell carcinoma tissues, respectively. A bright FITC signal was located mainly on the cell membrane and cytoplasm of tumor cells. In conclusion, the novel 12-residue peptide CSP-GD and 7-residue peptide CSP-KQ could specifically target human cervical cancer and may have the potential to be used in the diagnosis and targeted therapy of cervical cancer.
Keywords: Peptides; Tumor targeting; Cervical cancer; Phage display

Selenoproteins are a group of proteins which contain selenocysteine (Sec or U) in their primary structure. Selenoproteins play a critical role in antioxidant defense, hormone metabolism, immune responses and muscle development. The selenoprotein H (SELENOH) is essential in the regulation of gene expression in response to redox status and antioxidant defense. It has Sec residue located in conserved CXXU motif similar to other selenoproteins. However, exact biological function of Sec residue in SELENOH is not known in detail. Therefore, it is essential to understand the structural and functional role of Sec in SELENOH. In the present study, homology modelling and MD simulation were performed to understand the role of Sec residue in SELENOH. The modelled 3D structure of wild-SELENOH along with two mutants (Mut-U44C and Mut-41CS–SC44) was subjected to MD simulation. Based on simulation results, we demonstrate that wild-SELENOH structure is dynamically stabilized by network of intramolecular hydrogen bonding and internal residue contacts facilitated by Sec residue. In contrast, notable differences have been observed in residue contacts and stability in other two mutant structures. Additionally, docking studies revealed that 3PRGRKRK9 motif of wild-SELENOH interacts with HSE and STRE of DNA molecule as observed experimentally. Similar to earlier reports, our sequence analysis study pinpoints conserved 3PRGRKRK9 motif present in SELENOH perform dual role as AT-hook motif and NLS. Overall, the obtained results clearly illustrate Sec residue plays an important role to restore functionally active conformation of SELENOH. The present study broadened our current understanding regarding the role of selenocysteine in protein structure and function.
Keywords: Selenocysteine; Selenoprotein H; MD simulation; Molecular docking; DNA binding

Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides by Karol Sikora; Maciej Jaśkiewicz; Damian Neubauer; Marta Bauer; Sylwia Bartoszewska; Wioletta Barańska-Rybak; Wojciech Kamysz (609-619).
In view of an appreciable increase in resistance of Staphylococcus aureus to the conventional antibiotics, it is desired to develop new effective drugs. Antimicrobial peptides (AMPs) seem to be attractive candidates. In general, AMPs samples used for in vitro studies consist of a peptide, counter-ion, and water. The presence of the counter-ion could be significant as it affects peptide secondary structure and biological activity. The purpose of this study was to estimate the impact of counter-ion on antistaphylococcal activity of selected AMPs (CAMEL, citropin 1.1, LL-37, pexiganan, temporin A). To do this, three kinds of salts were prepared, namely, acetates, hydrochlorides, and trifluoroacetates. In addition, the hemolytic activity against human red blood cells (hRBCs) and cytotoxicity (HaCaT) were determined. The results indicate that there is a substantial difference between different salts, but the pattern is not consistent for the peptides. In general, the antistaphylococcal activity decreased in the order: CAMEL > temporin A > pexiganan > citropin 1.1 ≫ LL-37. The highest selectivity indexes were determined for CAMEL hydrochloride, pexiganan acetate, and temporin A trifluoroacetate. This study shows how important is to take into account the kind of counter-ions when designing novel peptide-based antimicrobials.
Keywords: Counter-ion; Antistaphylococcal activity; Antimicrobial peptides; CAMEL; Citropin 1.1; LL-37; Pexiganan; Temporin A

Milk is an important food for mammalian neonates, but its insufficient production is a nutritional problem for humans and other animals. Recent studies indicate that dietary supplementation with l-arginine (Arg) increases milk production in mammals, including sows, rabbits, and cows. However, the underlying molecular mechanisms remain largely unknown. The present study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that Arg enhances milk protein synthesis via activation of the mechanistic target of rapamycin (mTOR) cell signaling. PMECs were cultured for 4 days in Arg-free basal medium supplemented with 10, 50, 200, or 500 μmol/L Arg. Rates of protein synthesis and degradation in cells were determined with the use of l-[ring-2,4-3H]phenylalanine. Cell medium was analyzed for β-casein and α-lactalbumin, whereas cells were used for quantifying total and phosphorylated levels of mTOR, ribosomal protein S6 kinase (p70S6K), 4E-binding protein 1 (4EBP1), ubiquitin, and proteasome. Addition of 50–500 μmol/L Arg to culture medium increased (P < 0.05) the proliferation of PMECs and the synthesis of proteins (including β-casein and α-lactalbumin), while reducing the rates of proteolysis, in a dose-dependent manner. The phosphorylated levels of mTOR, p70S6K and 4EBP1 were elevated (P < 0.05), but the abundances of ubiquitin and proteasome were lower (P < 0.05), in PMECs supplemented with 200–500 μmol/L Arg, compared with 10–50 μmol/L Arg. These results provide a biochemical basis for the use of Arg to enhance milk production by sows and have important implications for improving lactation in other mammals (including humans and cows).
Keywords: Arginine; Mammary gland; Milk protein; mTOR signaling

Glycine enhances expression of adiponectin and IL-10 in 3T3-L1 adipocytes without affecting adipogenesis and lipolysis by Jingqing Chen; Xiaoshi Ma; Ying Yang; Zhaolai Dai; Zhenlong Wu; Guoyao Wu (629-640).
Glycine supplementation has been reported to enhance white-fat loss and improve sensitivity to insulin in animals with obesity or type 2 diabetes. However, the underlying mechanisms responsible for the beneficial effects of glycine remain largely unknown. The purpose of this study was to test the hypothesis that glycine regulates adipocyte differentiation, adipogenesis, and lipolysis, therefore, contributing to white-fat reduction. 3T3-L1 pre-adipocytes were induced to differentiate into adipocytes in the presence of glycine (0, 0.25, 1.0, and 2.0 mmol/L) or resveratrol (50 or 100 μmol/L, served as a positive control) during the differentiation process. Hela and HepG2 cells cultured with oleic acid to induce lipid accumulation in the presence of glycine (0, 1.0, and 2.0 mmol/L) or 10 μmol/L isoproterenol (served as a positive control) for 24 h. Intracellular lipid accumulation, intracellular triglycerides, lipid droplets’ diameters of mature adipocytes, mRNA, and protein levels of genes involved in the adipogenesis and lipolysis were analyzed. Isobutylxanthine–dexamethasone–insulin (MDI)-induced adipogenesis in 3T3-L1 cells were blocked by resveratrol, but not by glycine, as shown by decreased lipid contents, reduced diameters of lipid droplets, decreased protein abundances for peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), as well as increased protein abundance of peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), critical transcriptional factors that regulates adipogenesis. However, the mRNA levels of adiponectin and interleukin-10 (IL-10), two adipose-derived adipocytokines with anti-inflammatory effects, were greatly enhanced (P < 0.05) by 2 mmol/L glycine. Compared with non-treated controls, 10 μmol/L isoproterenol significantly decreased (P < 0.05) the intracellular lipid and triglyceride contents induced by oleic acid in Hela and HepG2 cells. mRNA level of fatty acid synthase (FASN), a gene involved in fatty acid synthesis, was significantly reduced (P < 0.05), while that for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase), genes involved in lipolysis were significantly enhanced (P < 0.05) by isoproterenol. However, oleic acid induced the accumulation of intracellular triglyceride and lipid contents were not affected by glycine. In conclusion, glycine exposure enhanced the mRNA levels of adipose-derived adiponectin and IL-10 without affecting adipogenesis and lipolysis in 3T3-L1 adipocytes. These findings provide a possible explanation for the anti-obesity and anti-diabetic effects of glycine that were previously reported in animal models. More studies are needed to uncover the underlying mechanisms responsible for this regulatory effect of glycine on anti-inflammatory adipocytokines expression in both in vitro and in vivo models.
Keywords: Glycine; Differentiation; Adipogenesis; Lipolysis; 3T3-L1; Adipocytokine

The target protein, Hcp1, was first described as part of the bacterial Type VI secretion system from Pseudomonas aeruginosa. The protein first self-assembles into a hexamer and then the hexamers further stack into a nanotubular structure. Hcp1 monomers were targeted for mutagenesis with two widely used photoactivatable amino acids: para-benzoyl phenylalanine or para-azidophenylalanine. The ability of these amino acids to form covalent adducts within the Hcp1 self-assembled system was investigated. Multiple residues, putatively of equal distance between the monomer–monomer interface were targeted. The efficiency of each amino acid to covalently link self-assembled hexamers was determined. The results demonstrate the choice and role of genetically encoded tools applied to complicated biological processes such as self-assembly and also suggested some structural dynamics of the Hcp-1 protein not obvious from crystallographic structures.
Keywords: Noncanonical amino acids; Self-assembly; Photochemistry

Fumarate adduction to cysteines has been implicated in the pathogenesis of several disorders. Its role, however, still remains elusive, and the need of predictive methods has not yet been met. The reactivity of cysteines found in fumarate-sensitive proteins was predicted when the collected data for eight network-type features were analyzed using classification models. Therefore, methods for evaluating the likelihood of a cysteine site to be modified by fumarate could be developed by combining concepts of network theory and machine learning.
Keywords: Protein succination; Cysteine reactivity; Fumarate; Residue interaction network