Short Amino Acid Sequences Research Articles

A Novel Cell Penetrating Peptide for the Differentiation of Human Neural Stem Cells.

Retinoic acid (RA) is a bioactive lipid that has been shown to promote neural stem cell differentiation. However, the highly hydrophobic molecule needs to first solubilize and translocate across the cell membrane in order to exert a biological response. The cell entry of RA can be aided by cell penetrating peptides (CPPs), which are short amino acid sequences that are able to carry bioactive cargo past the cell membrane. In this work, a novel cell penetrating peptide was developed to deliver RA to human neural stem cells and, subsequently, promote neuronal differentiation. The novel CPP consists of a repeating sequence, whose number of repeats is proportional to the efficiency of cell penetration. Using fluorescence microscopy, the mode of translocation was determined to be related to an endocytic pathway. The levels of β-III tubulin (Tubb3) and microtubule associated protein 2 (MAP2) expression in neural stem cells treated with RA conjugated to the CPP were assessed by quantitative immunocytochemistry.

Using multivariate mixed-effects selection models for analyzing batch-processed proteomics data with non-ignorable missingness.

In quantitative proteomics, mass tag labeling techniques have been widely adopted in mass spectrometry experiments. These techniques allow peptides (short amino acid sequences) and proteins from multiple samples of a batch being detected and quantified in a single experiment, and as such greatly improve the efficiency of protein profiling. However, the batch-processing of samples also results in severe batch effects and non-ignorable missing data occurring at the batch level. Motivated by the breast cancer proteomic data from the Clinical Proteomic Tumor Analysis Consortium, in this work, we developed two tailored multivariate MIxed-effects SElection models (mvMISE) to jointly analyze multiple correlated peptides/proteins in labeled proteomics data, considering the batch effects and the non-ignorable missingness. By taking a multivariate approach, we can borrow information across multiple peptides of the same protein or multiple proteins from the same biological pathway, and thus achieve better statistical efficiency and biological interpretation. These two different models account for different correlation structures among a group of peptides or proteins. Specifically, to model multiple peptides from the same protein, we employed a factor-analytic random effects structure to characterize the high and similar correlations among peptides. To model biological dependence among multiple proteins in a functional pathway, we introduced a graphical lasso penalty on the error precision matrix, and implemented an efficient algorithm based on the alternating direction method of multipliers. Simulations demonstrated the advantages of the proposed models. Applying the proposed methods to the motivating data set, we identified phosphoproteins and biological pathways that showed different activity patterns in triple negative breast tumors versus other breast tumors. The proposed methods can also be applied to other high-dimensional multivariate analyses based on clustered data with or without non-ignorable missingness.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

The long and the short of ceramides

The sphingolipid ceramide is not only a precursor of more complex sphingolipids, but also a potent signaling molecule. Specific ceramide species have distinct cellular functions, and each ceramide synthase therefore has particular roles in cells and organisms. Tidhar and colleagues, utilizing two ceramide synthases differing widely in fatty acid specificity, have identified a short amino acid sequence that is critical for this specificity. This work represents a crucial first step in the understanding of both the enzymology and the biology driving the diverse functions of ceramide.

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly.

Biology often provides the inspiration for functional soft matter, but biology can do more: it can provide the raw materials and mechanisms for hierarchical assembly. Biology uses polymers to perform various functions, and biologically derived polymers can serve as sustainable, self-assembling, and high-performance materials platforms for life-science applications. Biology employs enzymes for site-specific reactions that are used to both disassemble and assemble biopolymers both to and from component parts. By exploiting protein engineering methodologies, proteins can be modified to make them more susceptible to biology's native enzymatic activities. They can be engineered with fusion tags that provide (short sequences of amino acids at the C- and/or N- termini) that provide the accessible residues for the assembling enzymes to recognize and react with. This "biobased" fabrication not only allows biology's nanoscale components (i.e., proteins) to be engineered, but also provides the means to organize these components into the hierarchical structures that are prevalent in life.

Novel Dual Two‐Dimensional Liquid Chromatography Online Coupled to Ultraviolet Detector, Fluorescence Detector, Ion‐Trap Mass Spectrometer for Short Peptide Amino Acid Sequence Determination with Bottom‐Up Strategy

A novel dual two‐dimensional (2D) high‐performance liquid chromatography (LC) setup coupled online to an ultraviolet (UV) detector, fluorescence (FL) detector, and ion‐trap mass spectrometer (MS) has been developed for determining the amino acid sequence of short peptides using a novel bottom‐up strategy. Short peptides were electrothermally hydrolyzed to shorter peptides and amino acid enantiomers. The first 2D LC‐UV and FL system was used to separate and identify the produced parent and daughter short peptides and amino acid isomers and enantiomers in the hydrolysate; the second 2D LC‐MS was used to identify the presence of cysteine and obtain the molecular mass signals and N‐terminal peptide fragment ion signals for parent and daughter short peptides. The identified amino acid enantiomers are used to form any possible short peptides by permutation and combination in an order from dipeptide to a tripeptide, to a tetrapeptide, and to even higher short peptides. The correct short peptides are confirmed by comparing the molecular weights of the constituent amino acid enantiomers and the molecular weights of identified short peptides together, with the characteristic N‐terminal peptide fragment ion signals. The amino acid sequence of the dipeptide ester aspartame and the tripeptide glutathione was successfully determined by this method.

A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain

PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or “PB/LIE” free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

A Review of Protein Hydrolysates and Bioactive Peptides Deriving from Wastes Generated by Fish Processing

Fish wastes offer tremendous unexploited potential for value adding for such materials, collagen, gelatin, and hydrolysate. Additionally, fish waste may be suitable for the production of bioactive peptides that exhibit strong antihypertensive, antioxidative, and anticancer activities. This paper reviews the production of purified bioactive peptides from various fish waste protein hydrolysates via enzymatic hydrolysis after enzyme screening and optimization. The purification of bioactive peptides is carried out using ultra- and gel filtration as well as the RP-HPLC method. Purified peptide characterizations in terms of molecular weight, amino acid sequence, and composition are also provided, illustrating that peptides with low molecular weight and short amino acid sequences are more potent as bioactive peptides. Hence, further studies are encouraged to examine the bioactivity and bioavailability of protein peptides derived from fish wastes, especially those with anticancer characteristics.

Whey protein-derived peptide sensing by enteroendocrine cells compared with osteoblast-like cells: Role of peptide length and peptide composition, focussing on products of β-lactoglobulin hydrolysis

The short-term satiating effect of whey protein intake is higher than that obtained with other dietary proteins. The molecular basis underlying the secretagogue effect of whey proteins on the release of cholecystokinin, a gut hormone involved in appetite suppression, has not been deeply investigated. Using STC-1 enteroendocrine cells we analysed the effects of synthetic peptides and protein hydrolysates. The hypothesis that specific molecular features in terms of short amino acid sequences released after enzymatic digestion may exert a primary role in the cholecystokinin secretagogue activity of dietary proteins was not supported. However, the length of the peptide fragments derived from a protein during enzymatic digestion is relevant to this activity. Regarding the complexities of how food can interact with different cellular systems, our data suggest a novel biological activity of ALPMH, a known bioactive peptide product of β-lactoglobulin digestion, on cell growth and a related signalling pathway in osteoblast-like cells.

New chimeric peptide regulating insulin-like growth factor 1 regenerative effect for therapy in early stage osteoarthritis

Purpose: Osteoarthritis (OA) is the most frequent chronic musculoskeletal disease and knee OA is becoming the 4th most important global cause of disability in women and 8th in men. Early OA is estimated to affect nowadays more than 8.5 million of persons worldwide, this figure being estimated to reach 13.5 million in 2023. The 1st phase of osteoarthritis is the loss of cartilage. Due to his avascularity and his low number of stem cells, cartilage has a low mitotic activity and, consequently, cartilage has low ability to self-repair. Insulin-like growth factors-1 (IGF-I) is one of the most important growth factor and it’s involved in the regulation of chondrocyte metabolic function, as both a proliferative and differentiative factor. IGF-I is able to increase cell migration and also has an important function in an endocrine/paracrine and autocrine stimulating of matrix synthesis (proteoglycan and collagen type II). A new innovative chimeric peptide was developed to act on the balance of synthesis of IGF-I. This peptide combines a short amino acid sequence derived from growth hormone (GH) and an analog of somatostatin (SST). This two sequences, having an opposite effect on IGF-I releasing, induce a balanced regulation of IGF-I synthesis and its receptor expression (IGF-IR). We wanted to explore in vitro, on human chondrocytes cells culture, the effect of our chimeric peptide on stimulation of IGF-IR production as well as chondrocytes proliferation and differentiation. In addition, we evaluated his activity in in vivo spontaneous knee OA model. Methods: Human normal chondrocyte cell line was cultivated in cell growth medium with or without peptide. Cells were collected after 2, 4 and 8 days of culture. The total IGF-IR concentration was quantified using ELISA kit. The chondrogenic effect was observed by microscopy after staining with alcian blue to visualise proteoglycans. Five months old Duncan-Hartley guinea pigs (early OA) received one peptide or sham intraarticular injection per week for three weeks. Seven animals per group were used. Guinea pigs were sacrificed at 10 month old and both knees were collected for histology according OARSI-HISTOgp recommendations. Results: This GH-SST peptide increased the production of IGF-IR in human chondrocytes. The alcian blue staining revealed an increasing of cell clusters indicating a stimulation of chondrogenesis. In vivo test confirm the effect of this GH-SST peptide. Five months after injection, the quantitative histologic evaluation of knee guinea pig revealed that the GH-SST peptide injected group had a cartilage thickness significantly superior to the control group. Moreover, semiquantitative histologic scoring evaluation revealed a decrease of osteophytes development. Conclusions: This GH-SST peptide, by acting on IGF-I axis, improved the stimulation of chondrocyte cell growth and may be involved in the synthesis and maintenance of cartilage tissue. Indeed, this GH-SST peptide showed a long term effect to slow down tissues degeneration in knee OA model and preserved a better cartilage thickness. These results suggest that the GH-SST peptide may be an effective treatment of early articular cartilage defects.

A Particle Swarm Optimization-Based Approach with Local Search for Predicting Protein Folding.

The hydrophobic-polar (HP) model is commonly used for predicting protein folding structures and hydrophobic interactions. This study developed a particle swarm optimization (PSO)-based algorithm combined with local search algorithms; specifically, the high exploration PSO (HEPSO) algorithm (which can execute global search processes) was combined with three local search algorithms (hill-climbing algorithm, greedy algorithm, and Tabu table), yielding the proposed HE-L-PSO algorithm. By using 20 known protein structures, we evaluated the performance of the HE-L-PSO algorithm in predicting protein folding in the HP model. The proposed HE-L-PSO algorithm exhibited favorable performance in predicting both short and long amino acid sequences with high reproducibility and stability, compared with seven reported algorithms. The HE-L-PSO algorithm yielded optimal solutions for all predicted protein folding structures. All HE-L-PSO-predicted protein folding structures possessed a hydrophobic core that is similar to normal protein folding.

Minimized natural versions of fungal ribotoxins show improved active site plasticity

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins.

The effect of transglutaminase treatment on the physico-chemical properties of skim milk with added ethylenediaminetetraacetic acid

The influence of transglutaminase (TG) treatment on the changes in the physico-chemical properties of reconstituted skim milk (10 wt%) with different amounts of added ethylenediaminetetraacetic acid (EDTA) (0–50 mM) was studied. The partition of salts (Ca, Mg, K, Na and inorganic phosphate) was determined experimentally by atomic absorption and emission spectroscopy, colorimetry and 31P-nuclear magnetic resonance spectroscopy, and calculated with a model of the salt partition in milk based on equilibrium thermodynamics. Hydrodynamic radius, skim milk viscosity and casein micelle medium resolution substructure were measured by dynamic light scattering, rheology, and small-angle X-ray scattering (SAXS), respectively. TG treatment of milk did not influence the partition of salts upon the addition of EDTA as TG crosslinking is unlikely to disrupt the phosphate centre motifs on the short exon-encoded amino acid sequences on caseins. However, TG treatment prevented full dissociation of casein micelles when >15 mM EDTA was added, and induced higher viscosity increase than TG-untreated milk upon addition of EDTA. In accordance with the particle sizing results, SAXS demonstrated that TG treatment reduced the extent of EDTA-induced micellar disruption, as compared to milk without TG treatment.

Modulating Effect of Histidine on Short Arginine- and Tryptophan-Based Antimicrobial Peptides

Antimicrobial peptides (AMPs) are short sequences of aminoacids that organisms use as defense mechanism against foreign bodies. The selectivity of interaction with bacterial cells over mammalian cells underlines their significant potential in developing AMP-based drugs against antibiotics resistant bacteria. We started from a tryptophan- and arginine-rich peptide (RRWWRWWRR) which showed good antimicrobial efficacy. Seven new analogs were design by substitution of tryptophan or arginine residues with histidine residues.

Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor

Replication of the murine leukemia viruses is strongly suppressed in mouse embryonic stem (ES) cells. Proviral DNAs are formed normally but are then silenced by a large complex bound to DNA by the ES cell-specific zinc-finger protein ZFP809. We show here that ZFP809 expression is not regulated by transcription but rather by protein turnover: ZFP809 protein is stable in embryonic cells but highly unstable in differentiated cells. The protein is heavily modified by the accumulation of polyubiquitin chains in differentiated cells and stabilized by the proteasome inhibitor MG132. A short sequence of amino acids at the C terminus of ZFP809, including a single lysine residue (K391), is required for the rapid turnover of the protein. The silencing cofactor TRIM28 was found to promote the degradation of ZFP809 in differentiated cells. These findings suggest that the stem cell state is established not only by an unusual transcriptional profile but also by unusual regulation of protein levels through the proteasomal degradation pathway.

Neuropeptides and Addiction: An Introduction.

Neuropeptides are short sequences of amino acids that are coexpressed with neurotransmitters and which are widely expressed throughout the central nervous system. There is a large database of data pointing to critical roles for neuropeptides in modulating neurobiological responses to alcohol and drugs of abuse. Continued alcohol and drug use promote allostatic alterations in neuropeptide systems, and these changes contribute to excessive and uncontrolled intake that emerges with dependence. The neuropeptides that are reviewed in this chapter represent some of the most well-studied targets in the current drug and alcohol abuse literature. The goal of this chapter is to convey the significant roles that neuropeptides play in neurobiological responses to alcohol and drugs of abuse, and reinforce the idea that targeting neuropeptides and their receptors continue to be attractive avenues for treating drug and alcohol use disorders, as well as eating disorders.

Molecular engineering solutions for therapeutic peptide delivery.

Proteins and their interactions in and out of cells must be well-orchestrated for the healthy functioning and regulation of the body. Even the slightest disharmony can cause diseases. Therapeutic peptides are short amino acid sequences (generally considered <50 amino acids) that can naturally mimic the binding interfaces between proteins and thus, influence protein-protein interactions. Because of their fidelity of binding, peptides are a promising next generation of personalized medicines to reinstate biological harmony. Peptides as a group are highly selective, relatively safe, and biocompatible. However, they are also vulnerable to many in vivo pharmacologic barriers limiting their clinical translation. Current advances in molecular, chemical, and nanoparticle engineering are helping to overcome these previously insurmountable obstacles and improve the future of peptides as active and highly selective therapeutics. In this review, we focus on self-assembled vehicles as nanoparticles to carry and protect therapeutic peptides through this journey, and deliver them to the desired tissue.

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status.

Many neurodegenerative diseases arestill lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Oftenpotential new biomarkers do not make itinto the clinicdue to limitationsin their development andhigh costs.However,targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkersfor clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small moleculesin clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4). In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation. The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibratethe MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

Aminopeptidase P Mediated Targeting for Breast Tissue Specific Conjugate Delivery.

Cytotoxic chemotherapies are used to treat breast cancer, but are limited by systemic toxicity. The key to addressing this important issue is the development of a nontoxic, tissue selective, and molecular specific delivery system. In order to potentially increase the therapeutic index of clinical reagents, we designed an Aminopeptidase P (APaseP) targeting tissue-specific construct conjugated to a homing peptide for selective binding to human breast-derived cancer cells. Homing peptides are short amino acid sequences derived from phage display libraries that have the unique property of localizing to specific organs. Our molecular construct allows for tissue-specific drug delivery, by binding to APaseP in the vascular endothelium. The breast homing peptide evaluated in our studies is a cyclic nine-amino-acid peptide with the sequence CPGPEGAGC, referred to as PEGA. We show by confocal microscopy that the PEGA peptide and similar peptide conjugates distribute to human breast tissue xenograft specifically and evaluate the interaction with the membrane-bound proline-specific APaseP (KD = 723 ± 3 nM) by binding studies. To achieve intracellular breast cancer cell delivery, the incorporation of the Tat sequence, a cell-penetrating motif derived from HIV, was conjugated with the fluorescently labeled PEGA peptide sequence. Ultimately, tissue specific peptides and their conjugates can enhance drug delivery and treatment by their ability to discriminate between tissue types. Tissue specific conjugates as we have designed may be valuable tools for drug delivery and visualization, including the potential to treat breast cancer, while simultaneously minimizing systemic toxicity.

Force Sensitivity in Saccharomyces cerevisiae Flocculins

Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700pN and rupture lengths of up to 600nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.