Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells

Fluorophores are indispensable for imaging biological processes. We report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labelling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells.We thank FCT Portugal (Doctoral Fellowship, SFRH/BD/94779/2013 to F. M. F. S., Postdoctoral Fellowship, SFRH/BPD/103172/2014 to P. M. S. D. C.; projects PTDC/QUI-QUI/118315/2010 and PTDC/BBB BQB/0506/2012; PTDC/QEQ-QOR/1434/2014: PTDC/SAUFAR/119389/2010; FCT Investigator to G. J. L. B. and P. M. P. G.; iMed.ULisboa grant UID/DTP/04138/2013), EU (Marie-Curie CIG to G. J. L. B.; Marie-Sklodowska Curie ITN ProteinConjugates to G. J. L. B. and P. M. P. G.), DFG (SI 2117/1-1 to F. S.), CNPq Brazil (fellowship 200456/2015-6 to J. B. B.); Ministerio de Economía y Competitividad, Madrid, Spain (grant CTQ2014-54729-C2-1-P), Junta de Andalucía (grant P12-FQM-2140) and the EPSRC (G. J. L. B.) for financial support. G. J. L. B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt)

A Water-Bridged Cysteine-Cysteine Redox Regulation Mechanism in Bacterial Protein Tyrosine Phosphatases

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators.e thank CNPq Brazil (fellowship 200456/2015-6 to J.B.B. and grants 454507/2014-3 and 300606/2010-9 to H.T.), the Fundação para a Ciência e a Tecnologia (FCT Investigator award IF/00624/2015 to G.J.L.B.), the European Union (Marie-Sklodowska Curie Innovative Training Network Protein Conjugates; Marie Skłodowska-Curie Individual Fellowship 743640 to T.R.; Marie-Curie Intra-European Fellowship 626890 to O.B.), the Ministerio de Economía, Industria, y Competitividad (project CTQ2015-67727-R to F.C.), and the Biotechnology and Biological Sciences Research Council (PhD studentship to L.D.) for funding. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt, 676832 ). We also acknowledge funding by LISBOA-01-0145-FEDER-007391, co-financed by FEDER through the Programa Operacional Regional de Lisboa (Lisboa 2020) of PORTUGAL 2020 and by FCT Portugal

Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells

Fluorophores are indispensable for imaging biological processes. We report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labelling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells

Enhancement of the Anti-Aggregation Activity of a Molecular Chaperone Using a Rationally Designed Post-Translational Modification

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson's disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD