Time filter

Source Type

Hu W.,Sloan Kettering Cancer Center | Dooley J.,Vlaams Interuniversitair Instituut voor Biotechnologie | Dooley J.,Catholic University of Leuven | Chung S.S.,Sloan Kettering Cancer Center | And 9 more authors.
Blood | Year: 2015

Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types and to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1 bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, because miR-29b expression is unaltered in miR-29a/b-1- null HSCs, and only ectopic expression of miR-29a restoresHSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1 exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29 target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1-null HSCs. The loss of negative regulation of Dnmt3a by miR-29a is a major contributor to the miR-29a/b-1-null HSPC phenotype, as both in vitro Dnmt3a short hairpin RNA knockdown assays and a genetic haploinsufficiency model of Dnmt3a restored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1-deficient mice. Overall, these data demonstrate that miR-29a is critical for maintaining HSC function through its negative regulation of Dnmt3a. © 2015 by The American Society of Hematology.


Martinez-Rodriguez S.,University of Almeria | Garcia-Pino A.,Vrije Universiteit Brussel | Garcia-Pino A.,Vlaams Interuniversitair Instituut voor Biotechnologie | Heras-Vazquez F.J.L.,University of Almeria | And 6 more authors.
Journal of Bacteriology | Year: 2012

N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family. © 2012, American Society for Microbiology.


Azarkan M.,Free University of Colombia | Martinez-Rodriguez S.,Vrije Universiteit Brussel | Martinez-Rodriguez S.,Vlaams Interuniversitair Instituut Voor Biotechnologie | Martinez-Rodriguez S.,University of Almeria | And 5 more authors.
Journal of Biological Chemistry | Year: 2011

Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Van De Broek B.,IMEC | Van De Broek B.,Catholic University of Leuven | Devoogdt N.,Vlaams Interuniversitair Instituut voor Biotechnologie | Devoogdt N.,Vrije Universiteit Brussel | And 9 more authors.
ACS Nano | Year: 2011

Branched gold nanoparticles are potential photothermal therapy agents because of their large absorption cross section in the near-infrared window. Upon laser irradiation they produce enough heat to destroy tumor cells. In this work, branched gold nanoparticles are biofunctionalized with nanobodies, the smallest fully functional antigen-binding fragments evolved from the variable domain, the VHH, of a camel heavy chain-only antibody. These nanobodies bind to the HER2 antigen which is highly expressed on breast and ovarian cancer cells. Flow cytometric analysis and dark field images of HER2 positive SKOV3 cells incubated with anti-HER2 conjugated branched gold nanoparticles show specific cell targeting. Laser irradiation studies reveal that HER2 positive SKOV3 cells exposed to the anti-HER2 targeted branched gold nanoparticles are destroyed after five minutes of laser treatment at 38 W/cm 2 using a 690 nm continuous wave laser. Starting from a nanoparticle optical density of 4, cell death is observed, whereas the control samples, nanoparticles with anti-PSA nanobodies, nanoparticles only, and laser only, do not show any cell death. These results suggest that this new type of bioconjugated branched gold nanoparticles are effective antigen-targeted photothermal therapeutic agents for cancer treatment. © 2011 American Chemical Society.


Samyn D.R.,Linnaeus University | Ruiz-Pavon L.,Linnaeus University | Andersson M.R.,Linnaeus University | Popova Y.,Catholic University of Leuven | And 6 more authors.
Biochemical Journal | Year: 2012

In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symportmechanism, but alsomediates rapid activation of thePKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensionalmodel of Pho84 created on the basis of theGlpT permease, complemented with multiple sequence alignments, we selected Arg168 and Lys492, and Asp 178, Asp358 and Glu473 as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp358 (helix 7) and Lys492(helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp358 are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors. © The Authors Journal compilation © 2012 Biochemical Society.


Castro-Roa D.,Northumbria University | Garcia-Pino A.,Vrije Universiteit Brussel | Garcia-Pino A.,Vlaams Interuniversitair Instituut voor Biotechnologie | De Gieter S.,Vrije Universiteit Brussel | And 6 more authors.
Nature Chemical Biology | Year: 2013

Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities. © 2013 Nature America, Inc. All rights reserved.


Patent
Vlaams Interuniversitair Instituut Voor Biotechnologie | Date: 2011-08-12

The present invention relates to functional heavy chain antibodies, functional single domain heavy chain antibodies, functional VH domains, or functional fragments thereof comprising an amino acid which is neither a charged amino acid nor a C at position 45, and comprising an amino acid at position 103 independently chosen from the group consisting of R, G, K, S, Q, L, and P, and optionally an amino acid at position 108 independently chosen from the group consisting of Q, L and R, said positions determined according to the Kabat numbering.


Patent
Vlaams Interuniversitair Instituut Voor Biotechnologie and Life Sciences Partners | Date: 2011-03-16

The present invention relates to a use of VEGF-A for the manufacture of a medicament to treat any of frontotemporal lobe dementia, Alzheimers disease, Parkinsons disease, Huntingtons disease, and motor neuron disorders.


Patent
Vlaams Interuniversitair Instituut Voor Biotechnologie | Date: 2011-04-01

The present invention relates to functional heavy chain antibodies, functional single domain heavy chain antibodies, functional VH domains, or functional fragments thereof comprising an amino acid which is neither a charged amino acid nor a C at position 45, and comprising an amino acid at position 103 independently chosen from the group consisting of R, G, K, S, Q, L, and P, and optionally an amino acid at position 108 independently chosen from the group consisting of Q, L and R, said positions determined according to the Kabat numbering.


Patent
Vlaams Interuniversitair Instituut Voor Biotechnologie and Life Sciences Partners | Date: 2011-01-26

The present invention relates to a use of VEGF-B for the manufacture of a medicament to treat any of frontotemporal lobe dementia, Alzheimers disease, Parkinsons disease, Huntingtons disease, and motor neuron disorders.

Loading Vlaams Interuniversitair Instituut voor Biotechnologie collaborators
Loading Vlaams Interuniversitair Instituut voor Biotechnologie collaborators