S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates

We have systematically examined the S3 to S3′ subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3–S1 subsite requirements are more restricted than those of S1′–S3′. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg–Gly and Gly–Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.

Download Full-text

Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

Biochemical Journal ◽

10.1042/bj3470123 ◽

2000 ◽

Vol 347 (1) ◽

pp. 123-129 ◽

Cited By ~ 26

Author(s):

Fernada C. Vieira PORTARO ◽

Ana Beatriz F. SANTOS ◽

Maria Helena S. CEZARI ◽

Maria Aparecida JULIANO ◽

Luiz JULIANO ◽

...

Keyword(s):

Amino Acids ◽

Significant Influence ◽

Active Site ◽

Cathepsin B ◽

Cathepsin L ◽

Cysteine Proteinases ◽

Aminobenzoic Acid ◽

Site Analysis ◽

Hydrophobic Amino Acids ◽

Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Download Full-text

Biodegradable Polymers Derived from Amino Acids for Biological Applications

Advances in Science and Technology ◽

10.4028/www.scientific.net/ast.76.30 ◽

2010 ◽

Vol 76 ◽

pp. 30-35 ◽

Cited By ~ 3

Author(s):

Naomi Cohen-Arazi ◽

Ilanit Hagag ◽

Michal Kolitz ◽

Abraham J. Domb ◽

Jeoshua Katzhendler

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Ring Opening ◽

Building Blocks ◽

Ring Opening Polymerization ◽

Microwave Assisted Synthesis ◽

Hydroxy Acids ◽

Natural Amino Acids ◽

Direct Condensation ◽

Positively Charged

Optically active α-hydroxy acids derived from amino acids have been synthesized and polymerized into new biodegradable polyesters. The variety of functional side chains enables the design of positively charged, negatively charged, hydrophobic and hydrophilic chiral building blocks or any combination of these constituents. Hydroxy acids of 15 natural amino acids were prepared with retention of configuration using a straightforward and reliable method of diazotization of α-amino acids. Polyesters were synthesized from these hydroxy acids by a number of methods: direct condensation in bulk, microwave assisted synthesis and ring opening polymerization. The molecular weight of the prepared polymers ranges between 2000 to 5000Da for the direct condensation and the microwave methods, whereas the ring opening polymerization results in high molecular weight polymers (20000 to 30000Da). The polymers were analyzed for their optical activity (Circular Dichroism Spectroscopy), thermal properties (DSC), solubility, molecular weight and polydispersity (GPC), and aqueous degradation. These polymers were tested for their compatibility to neuronal cells growth and differentiation.

Download Full-text

Development of natural and unnatural amino acid delivery systems against hookworm infection

Precision Nanomedicine ◽

10.33218/prnano3(1).191210.1 ◽

2020 ◽

Vol 3 (1) ◽

pp. 471-482 ◽

Cited By ~ 3

Author(s):

Stacey Bartlett ◽

Mariusz Skwarczynski ◽

Xin Xie ◽

Istvan Toth ◽

Alex Loukas ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Cell Epitope ◽

Unnatural Amino Acid ◽

Nippostrongylus Brasiliensis ◽

Peptide Fragments ◽

Specific Pathogen ◽

Hydrophobic Amino Acids ◽

Natural Amino Acids

Peptide-based vaccines consist of short antigen fragments derived from a specific pathogen. Alone, these peptide fragments are poorly or non-immunogenic; however, when incorporated into a proper delivery system, they can trigger strong immune responses. To eliminate the need for toxic and often ineffective oral adjuvants, we designed single molecule-based self-adjuvating vaccines against hookworms using natural and unnatural hydrophobic amino acids. Two vaccine conjugates were synthesized, consisting of B-cell epitope p3, derived from the hookworm Na-APR-1 protein; universal T-helper peptide P25; and either double copies of unnatural lipoamino acid (2-amino-D,L-eicosanoic acid), or ten copies of the natural amino acid leucine. After challenge with the model hookworm, Nippostrongylus brasiliensis, mice orally immunized with the conjugates, but without adjuvant, generated antibody responses against the hookworm epitope, resulting in significantly reduced worm and egg burdens compared to control mice. We have demonstrated that vaccine nanoparticles composed exclusively of natural amino acids can be effective even when administered orally.

Download Full-text

Cathepsin K: a cysteine protease with unique kinin-degrading properties

Biochemical Journal ◽

10.1042/bj20040864 ◽

2004 ◽

Vol 383 (3) ◽

pp. 501-506 ◽

Cited By ~ 29

Author(s):

Emmanuel GODAT ◽

Fabien LECAILLE ◽

Claire DESMAZES ◽

Sophie DUCHÊNE ◽

Enrico WEIDAUER ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Smooth Muscles ◽

Cathepsin K ◽

Cysteine Proteases ◽

Unique Property ◽

Converting Enzyme ◽

Aminobenzoic Acid ◽

Wild Type ◽

Dose Dependent ◽

Type Receptor

Taking into account a previous report of an unidentified enzyme from macrophages acting as a kininase, the ability of cysteine proteases to degrade kinins has been investigated. Wild-type fibroblast lysates from mice, by contrast with cathepsin K-deficient lysates, hydrolysed BK (bradykinin), and released two metabolites, BK-(1–4) and BK-(5–9). Cathepsin K, but not cathepsins B, H, L and S, cleaved kinins at the Gly4–Phe5 bond and the bradykinin-mimicking substrate Abz (o-aminobenzoic acid)-RPPGFSPFR-3-NO2-Tyr (3-nitrotyrosine) more efficiently (pH 6.0: kcat/Km=12500 mM−1·s−1; pH 7.4: kcat/Km=6930 mM−1·s−1) than angiotensin-converting enzyme hydrolysed BK. Conversely Abz-RPPGFSPFR-3-NO2-Tyr was not cleaved by the Y67L (Tyr67→Leu)/L205A (Leu205→Ala) cathepsin K mutant, indicating that kinin degradation mostly depends on the S2 substrate specificity. Kininase activity was further evaluated on bronchial smooth muscles. BK, but not its metabolites BK(1-4) and BK(5-9), induced a dose-dependent contraction, which was abolished by Hoe140, a B2-type receptor antagonist. Cathepsin K impaired BK-dependent contraction of normal and chronic hypoxic rats, whereas cathepsins B and L did not. Taking together vasoactive properties of kinins and the potency of cathepsin K to modulate BK-dependent contraction of smooth muscles, the present data support the notion that cathepsin K may act as a kininase, a unique property among mammalian cysteine proteases.

Download Full-text

Amino Acid Dependent Performance of Modified Chitosan Against Bacteria and Red Blood Cell

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2021.19480 ◽

2021 ◽

Vol 21 (11) ◽

pp. 5443-5448

Author(s):

Xiaoyan Ju ◽

Lu Tian ◽

Xuantong Duan ◽

Zhuang Li ◽

Yongping Han ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Antibacterial Agents ◽

Molecular Design ◽

Antibacterial Properties ◽

E Coli ◽

Modified Chitosan ◽

Charged Amino Acids ◽

Hydrophobic Amino Acids ◽

Positively Charged

In order to combat antibiotic resistance, the development of new antibacterial agents is essential. In this study, we prepared four types of amino acid modified chitosan (CS-AA). Compared with chitosan modified with hydrophobic amino acids, the chitosan modified with positively charged amino acids showed higher antibacterial efficiency against Escherichia coli (E. coli) under similar grafting rate. CS-AA achieves antibacterial properties mainly by destroying the integrity of bacterial cell membranes. All the four types of CS-AA show low toxicity towards red blood cells. This work indicates that positively charged groups are more important than hydrophobic groups in the design of chitosan-based antibacterial agents, and provides helpful information for the molecular design of effective antibacterial agents.

Download Full-text

Comparative functional analysis of proteins containing low-complexity predicted amyloid regions

PeerJ ◽

10.7717/peerj.5823 ◽

2018 ◽

Vol 6 ◽

pp. e5823 ◽

Cited By ~ 2

Author(s):

Bandana Kumari ◽

Ravindra Kumar ◽

Vipin Chauhan ◽

Manish Kumar

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aromatic Amino Acids ◽

Low Complexity ◽

Amyloid Formation ◽

Charged Amino Acids ◽

Human Proteins ◽

Hydrophobic Amino Acids ◽

Compositional Pattern ◽

Positively Charged

Background In both prokaryotic and eukaryotic proteins, repeated occurrence of a single or a group of few amino acids are found. These regions are termed as low complexity regions (LCRs). It has been observed that amino acid bias in LCR is directly linked to their uncontrolled expansion and amyloid formation. But a comparative analysis of the behavior of LCR based on their constituent amino acids and their association with amyloidogenic propensity is not available. Methods Firstly we grouped all LCRs on the basis of their composition: homo-polymers, positively charged amino acids, negatively charged amino acids, polar amino acids and hydrophobic amino acids. We analyzed the compositional pattern of LCRs in each group and their propensity to form amyloids. The functional characteristics of proteins containing different groups of LCRs were explored using DAVID. In addition, we also analyzed the classes, pathways and functions of human proteins that form amyloids in LCRs. Results Among homopolymeric LCRs, the most common was Gln repeats. LCRs composed of repeats of Met and aromatic amino acids were amongst the least occurring. The results revealed that LCRs composed of negatively charged and polar amino acids were more common in comparison to LCRs formed by positively charged and hydrophobic amino acids. We also noted that generally proteins with LCRs were involved in transcription but those with Gly repeats were associated to translational activities. Our analysis suggests that proteins in which LCR is composed of hydrophobic residues are more prone toward amyloid formation. We also found that the human proteins with amyloid forming LCRs were generally involved in binding and catalytic activity. Discussion The presented analysis summarizes the most common and least occurring LCRs in proteins. Our results show that though repeats of Gln are the most abundant but Asn repeats make longest stretch of low complexity. The results showed that potential of LCRs to form amyloids varies with their amino acid composition.

Download Full-text

Positively Charged as Well as Hydrophobic Amino Acids in Orais’ Conserved N-Terminal Domain Contribute to Orai Function

Biophysical Journal ◽

10.1016/j.bpj.2009.12.3715 ◽

2010 ◽

Vol 98 (3) ◽

pp. 676a

Author(s):

Isabella Derler ◽

Barbara Lackner ◽

Judith Bergsmann ◽

Marc Fahrner ◽

Klaus Groschner ◽

...

Keyword(s):

Amino Acids ◽

Terminal Domain ◽

Hydrophobic Amino Acids ◽

Positively Charged

Download Full-text

Receptor properties of cyclic peptides composed of alternating natural amino acids and 3-aminobenzoic acid derivatives

Materials Science and Engineering C ◽

10.1016/s0928-4931(01)00380-0 ◽

2001 ◽

Vol 18 (1-2) ◽

pp. 125-133 ◽

Cited By ~ 8

Author(s):

Stefan Kubik ◽

Joachim Bitta ◽

Richard Goddard ◽

Daniela Kubik ◽

Susanne Pohl

Keyword(s):

Amino Acids ◽

Cyclic Peptides ◽

Aminobenzoic Acid ◽

Natural Amino Acids

Download Full-text

Probing cathepsin K activity with a selective substrate spanning its active site

Biochemical Journal ◽

10.1042/bj20030468 ◽

2003 ◽

Vol 375 (2) ◽

pp. 307-312 ◽

Cited By ~ 29

Author(s):

Fabien LECAILLE ◽

Enrico WEIDAUER ◽

Maria A. JULIANO ◽

Dieter BRÖMME ◽

Gilles LALMANACH

Keyword(s):

Wild Type Mouse ◽

Cathepsin K ◽

Cysteine Proteases ◽

Competitive Inhibitor ◽

Mouse Fibroblast ◽

Ionization State ◽

Proline Residue ◽

20 Nm ◽

S2 Subsite ◽

K Activity

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu67 which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2′ binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN2ph [where Abz represents o-aminobenzoic acid and EDN2ph (=EDDnp) represents N-(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly–Gly bond by cathepsin K (kcat/Km=426000 M−1·s−1). On the other hand, Abz-HPGGPQ-EDN2ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr67→Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN2ph to cathepsin K primarily depends on the S2 and S2′ subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN2ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN2ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.

Download Full-text