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1.
World J Microbiol Biotechnol ; 36(4): 53, 2020 Mar 14.
Article in English | MEDLINE | ID: mdl-32172335

ABSTRACT

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.


Subject(s)
Bacteria/enzymology , Fungi/enzymology , Protein Engineering/methods , Transglutaminases/metabolism , Bacterial Proteins/metabolism , Biocatalysis , Biotechnology , Fungal Proteins/metabolism , Immunoconjugates/metabolism , Substrate Specificity
2.
Peptides ; 102: 38-46, 2018 04.
Article in English | MEDLINE | ID: mdl-29486214

ABSTRACT

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides".


Subject(s)
Peptides/chemical synthesis , Pregnadienes/chemistry , Protein Aggregates , Solid-Phase Synthesis Techniques , Acylation , Amino Acid Sequence , Ethylamines/chemistry , Morpholines/chemistry , Peptides/chemistry , Peptides/genetics , Piperidines/chemistry , Pyridines/chemistry
3.
Cell Death Dis ; 5: e993, 2014 Jan 16.
Article in English | MEDLINE | ID: mdl-24434516

ABSTRACT

Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370-394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370-394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a K(D) of 12 µM. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370-394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370-394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection.


Subject(s)
Apoptosis Inducing Factor/metabolism , Apoptosis , Cyclophilin A/metabolism , Neurons/cytology , Neurons/metabolism , Oxidative Stress , Amino Acid Motifs , Apoptosis Inducing Factor/chemistry , Apoptosis Inducing Factor/genetics , Cyclophilin A/genetics , Down-Regulation , Humans , Mitochondria/enzymology , Mitochondria/metabolism , Neurons/enzymology , Protein Binding
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