Polypeptide Sequence Research Articles

Biomolecular condensates under extreme martian salt conditions.

Biomolecular condensates formed by liquid–liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming non-membraneous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the sub-seafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Membrane remodeling and vesicle formation by biomolecular condensates: A coarse grained simulation study.

Intrinsically disordered peptides (IDPs) can undergo liquid-liquid phase separation (LLPS) which is essential for membrane-less organelle formation and intracellular compartmentalization. Several experimental studies observed LLPS at or near the lipid membrane surface, leading to the alteration of the membrane morphology. Processes such as tubulation, endocytosis, exocytosis, and multilamellar membrane formation have also been observed in recent experiments. However, the molecular mechanisms and the governing molecular interactions remain unclear. Here we employ molecular dynamics simulations with different levels of coarse-graining to understand these long timescale processes. We study the coacervation of oppositely charged polypeptide sequences, namely, poly-glutamate (E30) and poly-lysine (K30) mixtures in the presence of lipid bilayers containing different ratios of POPC and POPG, with Martini 3.0 explicit solvent forcefield. We find that at least 20% anionic lipids are required to observe successful events of adsorption, followed by wetting, of the EK-coacervate on the bilayer. Upon wetting, the coacervate produces negative curvature to the bilayer and induces local demixing of lipids. Interestingly, the number of contacts between the polyelectrolytes decreases, unlike previous suggestions that predicted an increased overlap among the polyelectrolytes as the driving force of negative curvature formation. Next, we investigate the sensitivity of membrane deformation to the sequence (or charge patterns) of the IDPs. We observe that the IDPs with segregated charge patterns can remodel the membrane more prominently than IDPs with well-mixed charges. In order to understand the biologically relevant length- and time-scales, we developed a solvent-free coarse grained model for such systems. By tuning the IDP-lipid and IDP-IDP interactions, we observe endocytosis, exocytosis, and multilamellarity induced by the LLPS droplet into the bilayer vesicle.

Phosphomevalonate kinase regulates the MVA/MEP pathway in mango during ripening

Mango is a popular tropical fruit with a great diversity in taste and aroma, contributed primarily by terpenoids. Phosphomevalonate kinase (PMK) is a key enzyme for isoprenoid biosynthesis in the mevalonic acid (MVA) pathway responsible for terpenoids. In this study, two cultivars of mango, “Dashehari” and “Banganpalli”, showing opposite spatio-temporal patterns of ripening polarity, were investigated for studying the role of MiPMK in aroma production. MiPMK transcription and enzyme activity increased during ripening in both varieties. Expression in the early-ripening inner zones preceded that in the later-ripening outer zones of “Dashehari” while it was higher in the early ripening outer zones in “Banganpalli”. Polypeptide sequences of the two enzymes showed differences in a few amino acids that were also reflected in kinetic properties such as specific activity and pH optima. Silencing of MiPMK in “Dashehari” fruit by VIGS suppressed the kinase activity and led to changes in relative contributions of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways. This also altered the fruit metabolite profile with a reduction/disappearance of sesquiterpenes such as geranyl geraniol, trans-farnesol, β-caryophyllene, β-pinene, bisabolene and guaiane but the appearance of menthol and d-limonene in silenced fruit. The study shows that MiPMK levels may control downstream metabolite flux of the MVA pathway in mango.

Translation factor eIF5a is essential for IFNγ production and cell cycle regulation in primary CD8+ T lymphocytes

Control of mRNA translation adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Traditionally initiation of translation is considered to be a major translational control point, however, control of peptide elongation is also important. Here we show that the function of the elongation factor, eIF5a, is regulated dynamically in naïve CD8+ T cells upon activation by post-translational modification, whereupon it facilitates translation of specific subsets of proteins. eIF5a is essential for long-term survival of effector CD8+ T cells and sequencing of nascent polypeptides indicates that the production of proteins which regulate proliferation and key effector functions, particularly the production of IFNγ and less acutely TNF production and cytotoxicity, is dependent on the presence of functional eIF5a. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation contributes to post-transcriptional regulons important for the control of inflammation.

On the role of cell surface associated, mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae).

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (Frustule Associated Components), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC-MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins invivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.

PASylation improves pharmacokinetic of liposomes and attenuates anti-PEG IgM production: An alternative to PEGylation

PASylation, which was recently reported as the conjugation of pharmacologically active compounds with polypeptide sequences mainly made of proline, alanine and serine, has been proposed as an alternative to PEGylation. In this study, we designed PAS-modified liposomes (PASylated liposomes) and studied the effect of the incorporation of PAS-lipid on the stability and pharmacokinetic properties of liposomes, and compared them both in vitro and in vivo to PEGylated liposomes. Results showed that PASylated liposomes modified with single-chained PAS-lipid C16-(PA3)7 (SC-PAS-Lip) showed comparable storage and serum stability to PEGylated liposomes (PEG-Lip), and a significantly decreased macrophage uptake compared with unmodified liposomes. SC-PAS-Lip displayed long circulating pharmacokinetic profile which was not impacted by the repeated administration of liposomes, and they were less likely to induce the production of anti-PEG IgM compared with PEGylated liposomes, presenting PASylation as an alternative liposome modification strategy to PEGylation.

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria

BackgroundSome cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some β-sheets. MethodsThe software Kamal was used to scan the human reference proteome for short (7–11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of β-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. ResultsThousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a β-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. Conclusions: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. General SignificanceThis work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

(Invited) Biomolecular Engineering for Electrochemical Applications in Fuel Cells/Electrolyzers and Beyond

Control of ionomer thin film structures on metal surfaces is pivotal for efficient performance in electrochemical devices such as fuel cells and electrolyzers. Unfortunately, the assembly of these thin film structures is difficult to control using conventional methods and ideal arrangements remain unknown. Engineered polypeptides have emerged as powerful biomolecular tools in electrode assembly because binding sites and polypeptide structures can be easily modulated by changing the amino acid sequence. However, no studies have been conducted showing polypeptides can be engineered to interact with ionomers, attach them to metal surfaces, and control their arrangement. Our lab has recently developed this technology, using an elastin-like polypeptide to bind to metals, and bind to acidic and basic ionomer via ionic interactions. We use a quartz crystal microbalance with dissipation to provide detailed information about the loading, thickness, and binding behavior of the polypeptide and ionomer layers. We also use atomic force microscopy and grazing-incidence small-angle X-ray scattering to understand the impact of the biomolecules on ionomer phase separation. Finally, we have analyzed the performance (ionic conductivity) of the assembled films using interdigitated electrodes and electrochemical impedance spectroscopy. Through these techniques, we show that 1) our biomolecular system is highly flexible, easily adapting to different materials, 2) the polypeptide sequence can dictate ionomer phase separated structures, and 3) this system can be used to improve the performance of ionomer thin films and gain structure-function understanding. Generally, our results demonstrate that engineered polypeptides are promising tools for ionomer control in electrode engineering for fuel cells/electrolyzers and beyond. Furthermore, this work demonstrates that the creative combination of applied electrochemistry and biomolecular engineering supported and encouraged by leaders in each field can lead to unique research pathways that benefit heath, energy and the environment.

Repair of Alveolar Bone Defects with Osteogenic Polypeptide Modified HA_CF_PEEK Dental Implants

The implant implanted into the body directly interacts with the bone tissue around the implant through its surface. The surface properties of the implant can affect the adhesion, proliferation, and differentiation of surrounding cells and ultimately determine the quality of osseointegration and the effect of implantation surgery. In this project, nano-hydroxyapatite (n-HA) and carbon fiber (CF) were added into the matrix, poly(ether-ether-ketone) (PEEK), forming the composite material HA/CF/PEEK. Since biologically active proteins and peptides can promote cell viability and proliferation, the osteogenic polypeptide sequence BEE1 was polymerized and grafted onto the surface of HA/CF/PEEK composites to generate HA/CF/PEEK-DPP and HA/CF/PEEK-DPP-BEE1 composites. The above materials were tested for physical characterization and in vitro cell activity by establishing a Beagle dog dentition defect model. HA/CF/PEEK type composites were selected for peri-implant bone defect repair and compared with titanium mesh alone or Bio-Gide membrane for alveolar bone defect repair. In the experiment, the HA/CF/PEEK material modified with osteogenic polypeptides changed in element composition, hydrophilicity, and surface morphology. in vitro cell assays detected that the surface of HA/CF/PEEK material modified with osteogenic polypeptides was more conducive to cell adhesion and proliferation and had no cytotoxicity. The peri-implant defect repair test showed that the HA/CF/PEEK-DPP-BEE1 composite material for alveolar bone repair in experimental dogs was better than that of titanium mesh or Bio-Gide membrane. Meanwhile, the mineralization speed and histomorphometry test at the bone defect further proved that this material could better promote bone neogenesis at the defect. Mineralization.

Defective Human SRP Induces Protein Quality Control and Triggers Stress Response

Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

Transcription level and phylogeny analyses of Chlamydomonas reinhardtii arylsulfatases.

Sulfur is a required macroelement for all organisms, and sulfate deficiency causes growth and developmental defects. Arylsulfatases (ARS) hydrolyze sulfate from sulfate esters and make sulfate bioavailable for plant uptake. These enzymes are found in microorganisms and animals; however, plant genomes do not encode any ARS gene. Our database searches found nineteen ARS genes in the genome of Chlamydomonas reinhardtii. Among these, ARS1 and ARS2 were studied in the literature; however, the remaining seventeen gene models were not studied. Our results show that putative polypeptide sequences of the ARS gene models all have the sulfatase domain and sulfatase motifs found in known ARSs. Phylogenetic analyses show that C. reinhardtii proteins are in close branches with Volvox carterii proteins while they were clustered in a separate group from Homo sapiens and bacterial species (Pseudomonas aeruginosa and Rhodopirellula baltica SH1), except human Sulf1, Sulf2, and GNS are clustered with algal ARSs. RT-PCR analyses showed that transcription of ARS6, ARS7, ARS11, ARS12, ARS13, ARS17, and ARS19 increased under sulfate deficiency. However, this increase was not as high as the increase seen in ARS2. Since plant genomes do not encode any ARS gene, our results highlight the importance of microbial ARS genes.

Antifouling Electrochemical Biosensor Based on Conductive Hydrogel of DNA Scaffold for Ultrasensitive Detection of ATP

As an energy supplier, ATP plays an important role in various life activities, and there is an urgent need to develop an effective means of detecting ATP. However, the traditional sensors face serious nonspecific adsorption. In this work, an antifouling electrochemical biosensor based on the interpenetrating network of Y-DNA scaffold and polyaniline hydrogel was designed for ATP detection. The polyaniline hydrogel was conducive to the transport of electrons and ions, the structure of Y-DNA cross-linked by ATP aptamers in the polyaniline hydrogel achieved the effect of signal amplification. Super hydrophilic cellulose nanocrystals (CNCs) and zwitterion polypeptide sequence (Pep) were doped to play a synergistic antifouling effect. The hydrogel sensor we have built has a wide linear range of 0.1 pM-1 μM for ATP detection and a low detection limit of 0.025 pM (S/N = 3). For ATP detection in actual serum samples, the recovery of this sensor was 99.5%-106%, and the relative standard deviation was 0.4%-2.88%. It is proven that the sensor has good ATP detection performance, and it will provide a certain reference value for the detection of other biological small molecules.

Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato

Abstract. Effendy J. 2022. Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato. Biodiversitas 23: 4223-4233. Skinning injury is a major constrain in postharvest loss in sweetpotato due to the loss of skin from the surface of the roots. ASYMMETRIC LEAVES1/2 ENHANCER7(AE7) is an important cytosolic iron-sulfur (Fe-S) protein assembly (CIA) pathway. A partial cDNAIbI51 encoding AE7-like 1 was isolated from a skinning injury cDNA library to evaluate its potential role in response to skinning injury. The open reading frameof IbAE7-like 1 consisted of 324 nucleotides and the deduced polypeptide sequences consistedof 108 amino acids with missing 46 amino acid residues at 5? end. The amino acid sequence ofIbAE7-like 1 shared 100% identity toAE7-like 1 previously identified in Ipomoea triloba and Ipomoea nil and 89.91-100% identity from other plants species. The findings indicated that AE7-like 1 was highly conserved in Ipomoea sp. In addition to similarity to AE7-like 1, IbAE7-like 1 also showed similarities to MIP18 (89.91-93.52%); DUF59 (91.67%); FAM96B (90.74%); and proteins with unknown function (90.74-91.67%). IbAE7-like 1 mRNA transcripts showed transient expression in response to skinning injury. The phylogenetic analysis classified IbAE7-like 1 into three main classes. This study revealed IbAE7-like 1 may play a key role in plant defense and wound repair to prevent DNA damage due to skinning injuryin storage roots of sweetpotato.

Protein Vesicles with pH-Responsive Disassembly.

Protein biomaterials offer several advantages over those made from other components because their amino acid sequence can be precisely controlled with genetic engineering to produce a diverse set of material building blocks. In this work, three different elastin-like polypeptide (ELP) sequences were designed to synthesize pH-responsive protein vesicles. ELPs undergo a thermally induced hydrophobic transition that enables self-assembly of different kinds of protein biomaterials. The transition can be tuned by the composition of the guest residue, X, within the ELP pentapeptide repeat unit, VPGXG. When the guest residue is substituted with an ionizable amino acid, such as histidine, the ELP undergoes a pH-dependent hydrophobic phase transition. We used pH-responsive ELPs with different levels of histidine substitution, in combination with leucine zippers and globular, functional proteins, to fabricate protein vesicles. We demonstrate pH-dependent self-assembly, diameter, and disassembly of the vesicles using a combination of turbidimetry, dynamic light scattering, microscopy, and small angle X-ray scattering. As the ELP transition is dependent on the sequence, the vesicle properties also depend on the histidine content in the ELP building blocks. These results demonstrate the tunability of protein vesicles endowed with pH responsiveness, which expands their potential in drug-delivery applications.

β-Cyclodextrin-crosslinked synthetic neuropeptide Y-based nanosponges in epilepsy by contributing GABAergic signal

Neuropeptide Y (NPY) is a polypeptide sequence useful in regulating physiological functions like homeostasis, feeding, etc., but its usage is restricted due to its short half-life. β-cyclodextrin-crosslinked nanosponges improve the drug release and stability due to its wide cavity, which is helpful to deliver therapeutics. The present work aimed to formulate synthetic NPY-based nanocarriers as sponges by polymer condensation mechanism using design experiment to improve the peptide release and stability. The validated nanosponges exhibited a particle size of 423.42 ± 5.32 nm, 75.82 ± 7.43 % entrapment efficiency and 83.50 ± 6.54 % NPY release for 24 h. The NPY and β-cyclodextrin interaction was confirmed by X-ray diffraction, Fourier transform infrared and nuclear magnetic resonance spectroscopy. The NPY-loaded nanosponges were found stable for 6 months at two conditions (5 ± 2 °C and 25 ± 2 °C). The cross-linked nanocarriers of synthetic peptide-based nanosponges powder at different doses were administered intranasally using a metered-dose inhaler in the animal model to check its antiepileptic activity. The synthetic NPY-loaded nanosponges at higher doses showed significant antiepileptic effects equivalent to the standard drug (administered orally) in maximal electroshock and chemically-induced seizures with an increase of NPY in the brain directly proportional to GABAergic signalling by increase in GABA levels resulting in convulsions attenuation.

A CSDE1/Unr gene from Penaeus monodon: Molecular characterization, expression and association with tolerance to low salt stress

The RNA-containing cold shock domain binding protein E1 (CSDE1) has been found to have a key function in immunological stress regulation. In this work, a novel CSDE1 homologue, PmCSDE1, was identified in Penaeus monodon. The full length is 4251 bp, containing a 5’-UTR of 925 bp, a 3’-UTR of 323 bp, and a 31-base poly A tail. The ORF (Open reading frame) is 3003 bp and encodes 1000 amino acids. Its relative molecular mass is 110.91 kD, and its pI is 6.77. The PmCSDE1 polypeptide sequence has five CSP structural domains and one SUZ-C motif, which may have synergistic or retrograde functions in mRNA transcriptional regulation. The results of homology analysis and phylogenetic tree analysis showed that PmCSDE1 was most closely related to P. vannamei and clustered into one group (similarity up to 95.8%), and CSDE1 was highly conserved evolutionarily among different species. Quantitative analysis showed PmCSDE1 was widely expressed in all tissues examined. After low salt stress, PmCSDE1 was significantly downregulated in hepatopancreas and gill tissues. The knockdown of PmCSDE1 resulted in a significant increase in shrimp mortality. In addition, we screened two SNPs in the genome of PmCSDE1, PmCSDE1-In3-476 and PmCSDE1-E7-166, that may be significantly associated with low-salt tolerance by chi-square tests (χ2 (P) < 0.05). The findings are expected to provide a foundation for P. monodon resistance breeding research as well as preliminary data for the investigation of low-salt tolerant strains.

(Invited) Engineering Polypeptides for Controlled Assembly of Ionomer Thin Films: Expanding Beyond Typical Assembly Tools

Control of ionomer thin film structures on metal surfaces is pivotal for efficient performance in electrochemical devices such as fuel cells and electrolyzers. Unfortunately, the assembly of these thin film structures is difficult to control using conventional methods and ideal arrangements remain unknown. Engineered polypeptides have emerged as powerful biomolecular tools in electrode assembly because binding sites and polypeptide structures can be easily modulated by changing the amino acid sequence. However, no studies have been conducted showing polypeptides can be engineered to interact with ionomers, attach them to metal surfaces, and control their arrangement. Our lab has recently developed this technology, using an elastin-like polypeptide to bind to metals, and bind to acidic and basic ionomer via ionic interactions. We use a quartz crystal microbalance with dissipation to provide detailed information about the loading, thickness, and binding behavior of the polypeptide and ionomer layers. We also use atomic force microscopy and grazing-incidence small-angle X-ray scattering to understand the impact of the biomolecules on ionomer phase separation. Finally, we have analyzed the performance (ionic conductivity) of the assembled films using interdigitated electrodes and electrochemical impedance spectroscopy. Through these techniques, we show that 1) our biomolecular system is highly flexible, easily adapting to different materials, 2) the polypeptide sequence can dictate ionomer phase separated structures, and 3) this system can be used to improve the performance of ionomer thin films and gain structure-function understanding. Finally, alternative ionomer assembly techniques are discussed for future exploration. Generally, our results demonstrate that engineered polypeptides are promising tools for ionomer control in electrode engineering.

Controlling self-assembly of co-polypeptide by block ratio and block sequence

Block ratio and block sequence of polypeptide influenced the self-assembly of copolymer significantly. Tuning self-assembly to form various nanostructures with different properties through molecular design was a rather feasible strategy. Triblock copolymers based on poly( l -alanine) (PA), poly( l -valine) (PV) and poly (ethylene glycol) (PEG) with various block ratio and block sequence were synthesized. The number of amino acid residues was kept at 10. The sol-gel transition behavior, self-assemble nanostructure, secondary structure of the copolymers in aqueous solution were investigated to reveal that PA block between PEG and PV segment interfered the β-sheet structure, resulting in the reduced or increased sol-gel transition temperature or precipitation depended on the block ratio of PA and PV. Shorter PA or PV block displayed more significant influence on sol-gel transition. Interestingly, PV block between PEG and PA segment induced the formation of more stable β-sheet stacking structures, leading to sol-gel transition at higher temperature. PEG dehydration and the enhancement of ordered structure with temperature rising caused the sol-gel transition of these copolymers. We concluded herein that disordered structure in co-polypeptide can enhance thermo-sensitivity. • Conformation of synthesized non-natural polypeptides based on side-chain design. • Block sequence of non-natural polypeptides affected sol-gel transition. • Shorter peptide block influenced more significantly on sol-gel transition. • Less hydrophobic and weaker steric hindrance block at the tail enhanced the stability of nanostructures. • Disordered structure in co-polypeptide enhanced thermo-sensitivity.

A new approach for purification of the catalytic site of the angiotensin-conversion enzyme, N-domain, mediated by the ELP-Intein system

Angiotensin-converting enzyme I (ACE) is a key part of the renin-angiotensin system. Its main function is to regulate blood pressure and the balance of salts in the body. Somatic ACE has two domains, N-C-, each of which has a catalytic site that exhibits 60%sequence identity. The N-domain has a specific action in the hydrolysis of beta-amyloid bodies and angiotensin (1–7), which activates the MAS receptor and triggers anti-thrombotic and anti-inflammatory actions. Our goal was to obtain the catalytic site Ala361 to Gly468 of the N domain region, csACEN, without needing purification by chromatography. We employed a method that uses an Elastin-like Polypeptide (ELP) and Intein sequences linked to the peptide of interest. The more differential for obtaining the pure peptide was the cultivation temperatures in the synthesis of ELPcsACEN at 37 °C, with a significant increase in expression. In the purification by ELP precipitation, we recorded the highest efficiency in the concentrations of 0.57 M and 0.8 M of ammonium sulfate buffer. Intein autocleavage study allows removal of the ELP sequence at acidic pH, with the buffers MES and Tris-HCl The present study defined the best conditions for obtaining pure csACEN that the literature has not yet described for peptides. Obtaining pure csACEN aims at future studies for therapeutic use in hypertension, Alzheimer's, and oncology.

Antiangiogenic potential of small polypeptide sequences: In vivo assays, cytotoxicity, synthetic approaches and influence of C-terminal carboxyamidation

The search for new inhibitors of tyrosine kinase receptors involved in angiogenesis is a frequent topic in the design of antiangiogenic drugs. Michaloski and coworkers have obtained the peptide sequences PCAIWF-OH and PSAIWF-OH that showed potential inhibition of VEGF receptors. The present work describes the obtention of these peptides and their carboxyamidated analogues, which are prepared by a more efficient method. Their antiangiogenic activities were evaluated in vivo using the chicken embryo chorioallantoic membrane model. Assays with ARPE-19 cells were carried out to evaluate toxicity. PSAIWF-NH2 showed the higher inhibition of angiogenesis, at levels comparable to the bevacizumab. This carboxyamidated analogue carrying a serine has the advantage of not undergoing dimerization and it can be obtained by a more efficient and cheaper synthetic method. Retinal cells displayed in general high cell viability in the presence of the four peptides. PSAIWF-NH2 presented the highest angiogenic inhibition and the lowest toxicity. To evaluate the feasibility of prolonged and controlled release, PSAIWF-NH2 was incorporated into PLGA implants. The delivery profile was characterized by two phases, initially marked by a burst of release, followed by the sustained release of lower quantities during 36 days. Peptide concentrations were kept at levels that showed angiogenic inhibition.