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1.
Int J Biol Macromol ; 79: 903-12, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26071939

ABSTRACT

Hep1 is a mitochondrial Hsp70 (mtHsp70) co-chaperone that presents a zinc finger domain essential for its function. This co-chaperone acts to maintain mtHsp70 in its soluble and functional state. In this work, we have demonstrated that Leishmania braziliensis mtHsp70 (LbmtHsp70) is also dependent on the assistance of Hep1. To understand the L. braziliensis Hep1 (LbHep1) structure-function relationship, we produced LbHep1 and two truncated mutants corresponding to the C-terminal zinc finger domain and the N-terminal region. We observed that LbHep1 is composed of an unfolded N-terminal region and a ß-sheet-folded C-terminal domain, which holds the zinc-binding motif. Both LbHep1 and the zinc finger domain construction maintained LbmtHsp70 solubility in co-expression systems after cell lysis. In solution, LbHep1 behaved as a highly elongated monomer, probably due to the unfolded N-terminal region. Furthermore, we also observed that the zinc ion interacted with LbHep1 with high affinity and was critical for LbHep1 structure and stability because its removal from LbHep1 solutions altered the protein structure and stability. In vitro, LbHep1 protected, in sub-stoichiometric fashion, LbmtHsp70 from thermally induced aggregation but did not present intrinsic chaperone activity on model client proteins. Therefore, LbHep1 is a specific chaperone for LbmtHsp70.


Subject(s)
HSP70 Heat-Shock Proteins/chemistry , Mitochondrial Proteins/chemistry , Molecular Chaperones/chemistry , HSP70 Heat-Shock Proteins/metabolism , Leishmania braziliensis/chemistry , Mitochondria/chemistry , Mitochondria/genetics , Mitochondrial Proteins/genetics , Molecular Chaperones/genetics , Protein Binding , Protein Folding , Protein Structure, Secondary , Zinc Fingers/genetics
2.
J Biotechnol ; 173: 10-8, 2014 Mar 10.
Article in English | MEDLINE | ID: mdl-24417903

ABSTRACT

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.


Subject(s)
Cytoplasmic Dyneins/genetics , Gene Products, tat/genetics , Genetic Vectors , Peptides/metabolism , Recombinant Proteins/administration & dosage , Cytoplasmic Dyneins/metabolism , Gene Products, tat/metabolism , Gene Transfer Techniques , HeLa Cells , Humans , Lipids/pharmacology , Microscopy, Confocal , Microtubules/metabolism , Molecular Mimicry , Protamines/pharmacology , Recombinant Proteins/metabolism , Transfection
3.
Appl Microbiol Biotechnol ; 98(8): 3591-602, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24077724

ABSTRACT

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine™. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Gene Transfer Techniques , Biological Transport , Gene Expression , HeLa Cells , Humans , Models, Molecular , Plasmids , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Biochim Biophys Acta ; 1834(3): 697-707, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23298544

ABSTRACT

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.


Subject(s)
Bacterial Proteins/chemistry , Models, Molecular , Scattering, Small Angle , X-Ray Diffraction/methods , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Benzoates/chemistry , Benzoates/metabolism , Benzoates/pharmacology , Biofilms/drug effects , Biofilms/growth & development , Computer Simulation , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sorbic Acid/analogs & derivatives , Sorbic Acid/chemistry , Sorbic Acid/metabolism , Sorbic Acid/pharmacology , Xylella/genetics , Xylella/metabolism , Xylella/physiology
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