Bifunctional Peptide Research Articles

Extending Lifespan of Alzheimer’s Mode Nematode CL4176 Using a Novel Bifunctional Peptide with Inhibition of β-Amyloid Aggregation and Anti-oxidation

Alzheimer’s disease(AD) is a neurodegenerative disorder characterized by the deposition of β-amyloid peptide(Aβ) in the brain tissues, and an imbalance in the oxidant-antioxidant system. Compounds with antioxidant activity and the ability to inhibit Aβ aggregation therefore potentially treat AD. In this study, we designed an iron-porphyrin containing bifunctional peptide(BP, Deuterohemin-AlaHisThrValGluLysLeuProPhePheAsp) based on natural microperoxidase-11(MP-11) and typical β-sheet breaker LPFFD(iAβ5p). This BP substantially reduced the aggregation of Aβ and caused oligomer disassembly, by binding Aβ with affinity constant of 9.07 μmol/L. Furthermore, it showed a neuroprotective effect on Aβ25—35 induced toxicity in SH-SY5Y cells, and significantly alleviated Aβ-induced paralysis and extended lifespan in Aβ1—42 transgenic C. elegans(CL4176). It also showed a potent peroxidase activity of 20.8 U/mg, and scavenged free radicals both in vitro and in vivo. In addition, BP up-regulated the levels of hsp16.2, hsp16.41, and hsp12.6 mRNAs to 183.26%, 160.16%, and 162.64% respectively, and down-regulated that of hsp70 to 36.76% in C. elegans. Taken together, the synthetic BP inhibited Aβ aggregation and showed antioxidant activity, indicating the therapeutic potential of novel peptide drugs against AD.

Novel Bi-Functional 14-mer Peptides with Both Ovarian Carcinoma Cells Targeting and Magnetic Fe₃O₄Nanoparticles Affinity.

Fe3O4 magnetic nanoparticles (Fe3O4-MNPs) have attracted much interest for their potential medical applications due to their desirable magnetic properties. However, their potential cytotoxicity, high RES clearance in circulation, and nonspecific distribution in tissue might be the main obstacles in practice. In the present study, a novel bi-functional 14-mer peptide with both ovarian carcinoma cells targeting and magnetic Fe3O4 nanoparticles affinity was designed and synthesized, and then a facile and effective modification method was developed to bestow the Fe3O4-MNPs with tumor-targeting capability via modification, using the bi-functional peptides. First, on the basis of a tumor-targeting 7-mer peptide QQTNWSL (Q-L) and another Fe3O4-MNPs-targeting 7-mer peptide TVNFKLY (T-Y)—screened by phage-displayed peptide libraries—two bi-functional 14-mer peptides sequenced as LSWNTQQ-YLKFNVT (abbreviated as LQ-YT) and QQTNWSL-YLKFNVT (QL-YT) were synthesized through combining the Q-L peptide and T-Y peptide in predetermined configurations. Their specificity for bonding with A2780 tumor cells and affinity for Fe3O4-MNPs were verified. Then the bi-functional 14-mer peptides were applied to modify the Fe3O4-MNPs. Results showed that both bi-functional 14-mer peptides could be conjugated to the Fe3O4-MNPs surface with high affinity. Immunofluorescence and Prussian blue staining assays indicated that the LQ-YT-modified Fe3O4-MNPs could specifically bond to A2780 tumor cells. In addition to our findings suggesting that more β-turns and random coils are conducive to increasing polypeptide surface area for binding and exposing the target group and bonding sites on LQ-YT to external targets, we demonstrated that the bi-functional 14-mer peptide has affinity for Fe3O4-MNPs, and that Fe3O4-MNPs, which was modified with a 14-mer peptide, could be bestowed with a targeting affinity for ovarian carcinoma cells.

A genetically engineered Fc-binding amphiphilic polypeptide for congregating antibodies in vivo

We report herein an affinity-based hydrogel used in creating subcutaneous depots of antibodies in vivo. The biomaterials design centered on pG_EAK, a polypeptide we designed and expressed in E. coli. The sequence consists of a truncated protein G (pG) genetically fused with repeats of the amphiphilic sequence AEAEAKAK (“EAK”). Capture of IgG was demonstrated in vitro in gels prepared from admixing pG_EAK and EAK (“pG_EAK/EAK gel”). The binding affinities and kinetics of pG for IgG were recapitulated in the pG_EAK polypeptide. Injecting IgG antibodies formulated with pG_EAK/EAK gel into subcutaneous space resulted in retention of the antibodies at the site for at least six days, whereas only signal at background levels was detected in grafts injected with IgG formulated in saline or diffusion-driven gel. The local retention of IgG in pG_EAK/EAK gel was correlated with limited distribution of the antibody in liver, spleen and lymph nodes, in contrast to those injected with antibodies formulated in saline or non-Fc binding EAK gel. In addition, antibodies formulated with pG_EAK/EAK gel and injected in mouse footpads were found to retain at the site for 19 days. As a demonstration of potential bioengineering applications, thymic epithelial cells (TECs), the primary population of thymic stromal cells that are critical for the development of T-lymphocytes, were mixed with pG_EAK/EAK gel formulated with TEC-specific anti-EpCAM antibodies and injected subcutaneously into athymic nude mice. The injected TECs congregated into functional thymic units in vivo, supporting the development of both CD4+ and CD8+ T cells as well as Foxp3+ regulatory T cells in the mice. In conclusion, pG_EAK/EAK gel can be used to retain IgG locally in vivo, and can be tailored as scaffolds for controlling deposition of molecular and/or cellular therapeutics. Statement of significanceThe unique concept of the work centers on the genetic fusion of an Fc-binding domain and a self-assembling domain into a single polypeptide. To our knowledge, such bi-functional peptide has not been reported in the literature. The impact of the work lies in the ability to display IgG antibodies and Fc-fusion proteins of any specificity. The data shown demonstrate the platform can be used to localize IgG in vivo, and can be tailored for controlling deposition of primary thymic epithelial cells (TECs). The results support a biomaterials-based strategy by which TECs can be delivered as functional units to support T-lymphocyte development in vivo. The platform described in the study may serve as an important tool for immune engineering.

Erythroid-Progenitor-Targeted Gene Therapy Using Bifunctional TFR1 Ligand-Peptides in Human Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is a hereditary disease characterized by a deficiency in ferrochelatase (FECH) activity. FECH activity is responsible for the accumulation of protoporphyrin IX (PPIX). Without etiopathogenic treatment, EPP manifests as severe photosensitivity. 95% of affected individuals present a hypomorphic FECH allele trans to a loss-of-function (LOF) FECH mutation, resulting in a reduction in FECH activity in erythroblasts below a critical threshold. The hypomorphic allele promotes the use of a cryptic acceptor splice site, generating an aberrant FECH mRNA, which is responsible for the reduced level of wild-type FECH mRNA and, ultimately, FECH activity. We have previously identified an antisense oligonucleotide (AON), AON-V1 (V1), that redirects splicing to the physiological acceptor site and reduces the accumulation of PPIX. Here, we developed a specific strategy that uses transferrin receptor 1 (TRF1) as a Trojan horse to deliver V1 to erythroid progenitors. We designed a bifunctional peptide (P1-9R) including a TFR1-targeting peptide coupled to a nine-arginine cell-penetrating peptide (CPP) that facilitates the release of the AON from TFR1 in endosomal vesicles. We demonstrated that the P1-9R/V1 nanocomplex promotes the efficient and prolonged redirection of splicing towards the physiological splice site and subsequent normalization of WT FECH mRNA and protein levels. Finally, the P1-9R/V1 nanocomplex increases WT FECH mRNA production and significantly decreases PPIX accumulation in primary cultures of differentiating erythroid progenitors from an overt EPP-affected individual. P1-9R is a method designed to target erythroid progenitors and represents a potentially powerful tool for the invivo delivery of therapeutic DNA in many erythroid disorders.

Repeatedly Applied Peptide Film Kills Bacteria on Dental Implants

The rising use of titanium dental implants has increased the prevalence of peri-implant disease that shortens their useful life. A growing view of peri-implant disease suggests that plaque accumulation and microbiome dysbiogenesis trigger a host immune inflammatory response that destroys soft and hard tissues supporting the implant. The incidence of peri-implant disease is difficult to estimate, but with over 3 million implants placed in the USA alone, and the market growing by 500,000 implants/year, such extensive use demands additional interceptive approaches. We report a water-based, nonsurgical approach to address peri-implant disease using a bifunctional peptide film, which can be applied during initial implant placement and later reapplied to existing implants to reduce bacterial growth. Bifunctional peptides are based upon a titanium binding peptide (TiBP) optimally linked by a spacer peptide to an antimicrobial peptide (AMP). We show herein that dental implant surfaces covered with a bifunctional peptide film kill bacteria. Further, using a simple protocol for cleaning implant surfaces fouled by bacteria, the surface can be effectively recoated with TiBP-AMP to regain an antimicrobial state. Fouling, cleansing, and rebinding was confirmed for up to four cycles with minimal loss of binding efficacy. After fouling, rebinding with a water-based peptide film extends control over the oral microbiome composition, providing a novel nonsurgical treatment for dental implants.

Addition of free poloxamer 407 to a new gene vector P407-PEI-K12 solution forms a sustained-release in situ hypergel that enhances cell transfection and extends gene expression.

To address the concern around the efficiency/cytotoxicity ratio and the tumor-targeting effects of polyethylenimine (PEI), is a non-viral gene vector used for the delivery of the cancer therapy gene, poloxamer 407 (P407)-PEI-K12, was synthesized by cross-linking low-molecular weight PEI with P407 and further coupling a bifunctional peptide, K12, which is comprised of the tumor-targeting peptide tLyP-1 and the nuclear localization sequence. Furthermore, the addition of free P407 into the polymer/DNA complex solution produced a temperature-sensitive in situ gel-P407/P407-PEI-K12/DNA complex, which improved the effects of sustained-release gene delivery and transfection efficiency. The specificity, cytotoxicity and gene transfection efficiency of P407-PEI-K12 was investigated in Hela cells in vitro. The polymer efficiently prevented the degradation of plasmid DNA by DNase I and had a marked ability for serum tolerance. Agarose gel electrophoresis revealed that plasmid DNA was efficiently condensed and protected. The higher transfection efficiency of P407-PEI-K12h (the molar ratio of P407-PEI and K12 is 1:10) was achieved with a polymer and plasmid DNA ratio (w/w) of 20:1. The ability of free P407 to promote the transfection of the polymer/DNA complex was high (0.09%). The half-life of the P407/P407-PEI-K12-h/DNA gel complex was 228 min, and the transfection efficiency of the P407/P407-PEI-K12-h/DNA complex was markedly higher compared to that of the P407-PEI-K12-h/DNA complex at various release times.

Designer aromatic peptide amphiphiles for self-assembly and enzymatic display of proteins with morphology control.

We herein designed bi-functional aromatic peptide amphiphiles both self-assembling to fibrous nanomaterials and working as a substrate of microbial transglutaminase, leading to peptidyl scaffolds with different morphologies that can be enzymatically post-functionalized with proteins.

SPIND: a reference-based auto-indexing algorithm for sparse serial crystallography data.

SPIND (sparse-pattern indexing) is an auto-indexing algorithm for sparse snapshot diffraction patterns ('stills') that requires the positions of only five Bragg peaks in a single pattern, when provided with unit-cell parameters. The capability of SPIND is demonstrated for the orientation determination of sparse diffraction patterns using simulated data from microcrystals of a small inorganic molecule containing three iodines, 5-amino-2,4,6-triiodoisophthalic acid monohydrate (I3C) [Beck & Sheldrick (2008 ▸), Acta Cryst. E64, o1286], which is challenging for commonly used indexing algorithms. SPIND, integrated with CrystFEL [White et al. (2012 ▸), J. Appl. Cryst. 45, 335-341], is then shown to improve the indexing rate and quality of merged serial femtosecond crystallography data from two membrane proteins, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2 and the NTQ chloride-pumping rhodopsin (CIR). The study demonstrates the suitability of SPIND for indexing sparse inorganic crystal data with smaller unit cells, and for improving the quality of serial femtosecond protein crystallography data, significantly reducing the amount of sample and beam time required by making better use of limited data sets. SPIND is written in Python and is publicly available under the GNU General Public License from https://github.com/LiuLab-CSRC/SPIND.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy

Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.

Bifunctional peptide hybrids targeting the matrix of mitochondria

The mitochondrial organelle is associated with many diseases, including diabetes, age-related neuro-degenerative diseases and cancer. Therefore, the effective delivery of drug molecules to mitochondria became increasingly important during the past years. Within this work, we designed and analyzed bifunctional hybrid peptides comprised of a mitochondrial targeting sequence (MTS) attached to a cell-penetrating peptide (CPP). Our results demonstrate that choice of the MTS must be carefully undertaken, since not every MTS that was selected was comparably capable to target mitochondria. In addition, we highlight the use of the CPP sC18 as necessary part of the hybrid construct, inducing not only cellular uptake, but likewise supporting sub-organelle uptake into the mitochondrial matrix. The herein designed cell-permeable mitochondrial targeting peptide was furthermore proven to enhance the intracellular uptake of the cytostatic drug chlorambucil, making it a powerful candidate for further studies in this important field.

Bifunctional anticaries peptides with antibacterial and remineralizing effects.

Initial dental caries often occurs in clinic. Reduction of cariogenic bacteria and promotion of remineralization are effective ways to control them. This study was to develop bifunctional anticaries peptides with antibacterial and remineralizing properties. We designed peptides TDH19, TNH19, and TVH19 and selected one through comparing their minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) against Streptococcus mutans and their reaction on mineralization. Then the bifunction of the selected peptide was studied through: (a) effects on S.mutans biofilm, (b) remineralizing effects on initial lesions and (c) stability in saliva and cytocompatibility to human oral keratinocytes (HOKs). TVH19 showed the lowest MIC and MBC and a better mineralizing ability. It inhibited new biofilm formation and reduced the viability of old biofilm (p<0.05). Treating initial caries with TVH19 led to greater recovery of surface microhardness, shallower lesion depth, and higher mineral content (p<0.05). No significant differences were observed between TVH19 and NaF samples (p>0.05). TVH19 was stable in saliva and had little effect on HOKs. The novel bifunctional anticaries peptide TVH19 was developed with remarkable antibacterial activity and the potential to enhance remineralization of initial caries.

99mTc][Tc(N)PNP43]-Labeled RGD Peptides As New Probes for a Selective Detection of αvβ3 Integrin: Synthesis, Structure-Activity and Pharmacokinetic Studies.

New integrin-selective molecules suitable for therapeutic or imaging purposes are currently of interest in development of effective personalized medical platforms. RGDechi is a bifunctional peptide selective for integrin αvβ3. Herein, RGDechi and three truncated derivatives functionalized with a cysteine (1-4) were synthesized and labeled with the [99mTc][Tc(N)PNP43]-synthon ([PNP43 = (CH3)2P(CH2)2N(C2H4OCH3)(CH2)2P(CH3)2]) (99mTc1-4) as a basis for selective integrin recognition. The pharmacological parameters of all radiolabeled peptides were assessed along with the pharmacokinetic profiles of the most promising 99mTc1 and 99mTc2 compounds both on healthy and melanoma-bearing mice. Their metabolism and metabolite identification are also reported. 99mTc1-2 are able to discriminate between endogenously expressed integrins αvβ3 and αvβ5 and possess favorable pharmacokinetics characterized by low liver uptake and rapid elimination from nontarget tissues resulting in positive target-to-nontarget ratios. Results are encouraging; the presented construct can be considered the starting point for the development of agents for the selective detection of αvβ3 expression by SPECT.

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature.

To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

Functionalization of stable fluorescent nanodiamonds towards reliable detection of biomarkers for Alzheimer\u2019s disease

BackgroundStable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery.ResultsWe report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long β-sheet breaker peptide that is able to recognize amyloid β (Aβ) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains.ConclusionsThe fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used tostain Aβ aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aβ aggregates involved in the pathogenesis of the Alzheimer disease.

A One-Pot "Triple-C" Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides.

A broadly applicable one-pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)-catalyzed reaction (CEPS/CLIPS/CuAAC; "triple-C") locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head-to-tail by using the peptiligase variant omniligase-1 (CEPS). Subsequent ligation-cyclization with tetravalent (T41/2 ) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF-T41/2 ) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero-bifunctional variants (UF/FU-T41/2 ), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the "bifunctional tetracyclic peptide" inhibitor concept.

Loop Replacement Enhances the Ancestral Antibacterial Function of a Bifunctional Scorpion Toxin.

On the basis of the evolutionary relationship between scorpion toxins targeting K+ channels (KTxs) and antibacterial defensins (Zhu S., Peigneur S., Gao B., Umetsu Y., Ohki S., Tytgat J. Experimental conversion of a defensin into a neurotoxin: Implications for origin of toxic function. Mol. Biol. Evol. 2014, 31, 546–559), we performed protein engineering experiments to modify a bifunctional KTx (i.e., weak inhibitory activities on both K+ channels and bacteria) via substituting its carboxyl loop with the structurally equivalent loop of contemporary defensins. As expected, the engineered peptide (named MeuTXKα3-KFGGI) remarkably improved the antibacterial activity, particularly on some Gram-positive bacteria, including several antibiotic-resistant opportunistic pathogens. Compared with the unmodified toxin, its antibacterial spectrum also enlarged. Our work provides a new method to enhance the antibacterial activity of bifunctional scorpion venom peptides, which might be useful in engineering other proteins with an ancestral activity.

Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

BackgroundChimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. However, excessive activity and poor control over such engineered T cells cause significant safety challenges, such as cytokine release syndrome and organ toxicities. To enhance the specificity and controllable activity of CAR-T cells, we report a novel switchable dual-receptor CAR-engineered T (sdCAR-T) cell and a new switch molecule of FITC-HM-3 bifunctional molecule (FHBM) in this study.MethodsWe designed a fusion molecule comprising FITC and HM-3. HM-3, an antitumor peptide including an Arg-Gly-Asp sequence, can specifically target integrin αvβ3 that is presented on some tumor cells. Moreover, to improve the specificity of CAR-T cells, we also generated the sdCAR-T cell line against cognate tumor cells expressing human mesothelin (MSLN) and integrin αvβ3. Finally, the activity of sdCAR-T cell and FHBM is verified via in vitro and in vivo experiments.ResultsIn the presence of FHBM, the designed sdCAR-T cells exerted high activity including activation and proliferation and had specific cytotoxicity in a time- and dose-dependent manner in vitro. Furthermore, using a combination of FHBM in nude mice, sdCAR-T cells significantly inhibited the growth of MSLN+ K562 cells and released lower levels of the cytokines (e.g., interleukin-2, interferon γ, interleukin-6, and tumor necrosis factor α) relative to conventional CAR-T cells, obtaining specific, controllable, and enhanced cytotoxicity.ConclusionsOur data indicate that FHBM can accurately control timing and dose of injected CAR-T cells, and sdCAR-T cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvβ3. Therefore, combination therapies using sdCAR-T cells and the switch molecule FHBM have significant potential to treat malignancies.

Conjugates of Cell Adhesion Peptides for Therapeutics and Diagnostics Against Cancer and Autoimmune Diseases.

Overexpressed cell-surface receptors are hallmarks of many disease states and are often used as markers for targeting diseased cells over healthy counterparts. Cell adhesion peptides, which are often derived from interacting regions of these receptor-ligand proteins, mimic surfaces of intact proteins and, thus, have been studied as targeting agents for various payloads to certain cell targets for cancers and autoimmune diseases. Because many cytotoxic agents in the free form are often harmful to healthy cells, the use of cell adhesion peptides in targeting their delivery to diseased cells has been studied to potentially reduce required effective doses and associated harmful side-effects. In this review, multiple cell adhesion peptides from extracellular matrix and ICAM proteins were used to selectively direct drug payloads, signal-inhibitor peptides, and diagnostic molecules, to diseased cells over normal counterparts. RGD constructs have been used to improve the selectivity and efficacy of diagnostic and drug-peptide conjugates against cancer cells. From this precedent, novel conjugates of antigenic and cell adhesion peptides, called Bifunctional Peptide Inhibitors (BPIs), have been designed to selectively regulate immune cells and suppress harmful inflammatory responses in autoimmune diseases. Similar peptide conjugations with imaging agents have delivered promising diagnostic methods in animal models of rheumatoid arthritis. BPIs have also been shown to generate immune tolerance and suppress autoimmune diseases in animal models of type-1 diabetes, rheumatoid arthritis, and multiple sclerosis. Collectively, these studies show the potential of cell adhesion peptides in improving the delivery of drugs and diagnostic agents to diseased cells in clinical settings.

Recent Advances in Antibacterial and Antiendotoxic Peptides or Proteins from Marine Resources.

Infectious diseases caused by Gram-negative bacteria and sepsis induced by lipopolysaccharide (LPS) pose a major threat to humans and animals and cause millions of deaths each year. Marine organisms are a valuable resource library of bioactive products with huge medicinal potential. Among them, antibacterial and antiendotoxic peptides or proteins, which are composed of metabolically tolerable residues, are present in many marine species, including marine vertebrates, invertebrates and microorganisms. A lot of studies have reported that these marine peptides and proteins or their derivatives exhibit potent antibacterial activity and antiendotoxic activity in vitro and in vivo. However, their categories, heterologous expression in microorganisms, physicochemical factors affecting peptide or protein interactions with bacterial LPS and LPS-neutralizing mechanism are not well known. In this review, we highlight the characteristics and anti-infective activity of bifunctional peptides or proteins from marine resources as well as the challenges and strategies for further study.

Autonomous Synthesis of Fluorescent Silica Biodots Using Engineered Fusion Proteins.

Formation of biological materials is a well-controlled process that is orchestrated by biomolecules such as proteins. Proteins can control the nucleation and mineralization of biomaterials, thereby forming the hard tissues of biological organisms, such as bones, teeth, and shells. In this study, the design and implementation of multifunctional designer proteins are demonstrated for fluorescent silica micro/nanoparticle synthesis. The R5 motif of silaffin polypeptide, which is known for its silicification capability, was fused genetically into three spectrally distinct fluorescent proteins with the intention of forming modified fluorescent proteins. The bifunctional R5 peptide domain served as a tag to provide silica synthesis at ambient conditions. Three functional fusion constructs have been prepared, including GFPmut3-R5, Venus YFP-R5, and mCherry-R5. Recombinant fluorescent proteins were purified using silica-binding peptide tag through silica gel resin. Purified proteins were tested for their binding affinity to silica using quartz crystal microbalance with dissipation monitoring to make sure they can interact strong enough with the silica surfaces. Later, engineered fluorescent proteins were used to synthesize silica nano/microparticles using silica precursor materials. Synthesized silica particles were investigated for their fluorescence properties, including time-resolved fluorescence. Additionally, elemental analysis of the particles was carried out using electron energy loss spectroscopy and energy-filtered transmission electron microscopy. Last, they were tested for their biocompatibility. In this study, we aimed to provide a biomimetic route to synthesize fluorescent silica nanoparticles. Recombinant fluorescent proteins-directed silica nanoparticles synthesis offers a one-step, reliable method to produce fluorescent particles both for biomaterial applications and other nanotechnology applications.