WHO I AM

CURIOUS, PERSISTENT AND TEAM PLAYER

I am a young researcher in the Extremophiles and Large Molecular assemblies group
I have always been truly fascinated by the limits of Life and by the extraordinary adaptations of the organisms that thrives in extreme environment. I am also interested in the world of large peptidases that, although essential for the cell, are still largely unknown.

During my PhD, I had the great opportunity to work in the multicultural and multidisciplinary environment that is the IBS, which is part of the PSB (Partnership for Structural Biology). It allowed me to get hands on experience on various technics and to meet and discuss with people from very different backgrounds.

I have always been curious, that may be why I choose this field of work. These years of research helped me to develop my perseverance, keeping in mind the aim of the project.

MY RESEARCH

During my PhD, under the supervision of the Dr Bruno Franzetti and the Dr Frank Gabel, I have been interested in the further characterization of a family of aminopeptidase forming 12 subunits complexes called TET. I used an integrated strategy to get insight into the oligomerization-function relationship of this aminopeptidase family using the Archaea Pyrococcus horikoshii as a model.

Throughout this project, I was able to get hands-on experience on biochemistry, enzymology, phylogenomic and various biophysical technics used as an integrated strategy for protein characterization.

I am now the project manager for the development of a start-up project : DEEP BLUE PROCESS.

LARGE INTRACELLULAR PEPTIDASES

Efficient and controlled intracellular polypeptide breakdown is a primordial requirement that controls many cellular processes. And yet, the role of the abundant peptidases present in the cytosol is still largely unknown.

A proteomic study of cells extract from a halophilic organism under stress led to the discovery of TET, a 12-subunits aminopeptidase forming tetrahedral shaped complexes. The physiological role of TET peptidases is still unclear but their high evolutionary conservation in the three kingdoms of life suggests that they fulfil important biological functions.

This hypothesis is supported by recent work on the eukaryotic tetrahedral aminopeptidase DNPEP that has been proposed to be a key player in the central nervous system.

MY PUBLICATIONS

Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation

TET aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model.

Point mutations allowed the capture of a stable, catalytically active precursor. Small angle X-ray scattering (SAXS) revealed that it is a dimer whose architecture in solution is identical to that determined by X-ray crystallography within the fully assembled TET particle. SAXS also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates.

Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.

Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques.

Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates.

This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties

TET aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model.

In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When co-expressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analyzed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles.

Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

TET peptidases: A family of tetrahedral complexes conserved in prokaryotes.

The TET peptidases are large polypeptide destruction machines present among prokaryotes. They form 12-subunits hollow tetrahedral particles, and belong to the family of M42 metallo-peptidases.

Structural characterization of various archaeal and bacterial complexes has revealed a unique mechanism of internal compartmentalization and peptide trafficking that distinguishes them from the other oligomeric peptidases. Different versions of the TET complex often co-exist in the cytosol of microorganisms. In depth enzymatic studies have revealed that they are non-processive cobalt-activated aminopeptidases and display contrasting substrate specificities based on the properties of the catalytic chambers. Recent studies have shed light on the assembly mechanism of homo and hetero-dodecameric TET complexes and shown that the activity of TET aminopeptidase towards polypeptides is coupled with its assembly process. These findings suggested a functional regulation based on oligomerization control in vivo.

This review describes a current knowledge on M42 TET peptidases biochemistry and discuss their possible physiological roles. This article is a part of the Special Issue entitled: «A potpourri of proteases and inhibitors: from molecular toolboxes to signalling scissors».

MEETINGS &
CONFERENCES

  • 2emes Journées du GT ARCHAEA

    20 Novembre 2012 GRENOBLE, FRANCE

    15 min oral presentation – plenary session "Role and mode of action of a family of ATP independent peptidases complexes in Archaea."

  • SFBBM-SFB 2012 congress “From molecular mechanisms to integrated life processes”

    21-23 November 2012 GRENOBLE, FRANCE

    Poster

  • Molecular Biology of Archaea 4 – mboa4

    19/05/2014 au 22/05/2014 Paris, France

    20 min oral presentation – plenary session "Pyrococcus horikoshii TET aminopeptidases can form multi-subunit peptidasome: distribution, assembly process and functional regulation."

  • CAREX Conference on Life in Extreme Environments

    18-20 October 2011 Dublin, Ireland

    15 min oral presentation – split session "Bioprospecting for novel proteases in deep-sea hyperthermophilic Pyrococcales"

  • Young Scientist Forum 2013

    3-6 July 2013 Saint-Petersburg, Russia

    Poster

  • Federation of European Biochemical Societies CONGRESS 2013 "Mechanisms in Biology"

    July 6th – 11th 2013Saint-Petersburg, Russia

    Poster

  • 6ème colloque "Protéolyse Cellulaire" de la Société Française de Biochimie et Biologie Moléculaire

    28 au 30 Nov 2012Clermont-Ferrand, France

    20 min oral presentation – plenary session "Deciphering the role of large ATP-independent peptidase complexes in extremophilic Archaea."

  • 14h International meeting on thermophile biology

    August 27th - September 1st 2017 Skukuza conference center, Mpumalanga, South africa

    20 min oral presentation – plenary session "Integrated bio-structural strategy unravels the assembly and activation of a giant aminopeptidase"

MY RESUME

Skills

Project Research Management

Project development, task planning, progress management

Biochemistry

Enzymology, protein chromatography (FPLC, HPLC, PAGE)

Biophysics

Small Angle X-ray/Neutron Scattering (SANS/SAXS), Analytical Ultracentrifugation (AUC), Crystallography

Molecular biology

Expression cloning, gel electrophoresis, macromolecular blotting and probing

Structural biology

Crystallization, X-ray crystallography, analyzing structure

Genetic

Extraction and purification of DNA/RNA, qRT-PCR, micro-array

Education

2010 - 2014

PhD Thesis in Biochemistry, University Joseph Fourier, at the Institut de Biologie Structurale in Grenoble, France, in the Extremophiles and Large Molecular Assemblies group

2008 - 2010

Master’s degree, major in Molecular and Cellular Biology, specialization: Genetics – option: RNA and Evolution – at the University Pierre et Marie Curie (University Paris VI) – with honors

March 2011

Higher European Research Course for Users of Large Experimental Systems (HERCULES) at the Grenoble Innovation for Advanced New Technologies (GIANT) campus in Grenoble, France

August 2008

European Intensive program on Origins of life and life in space at the University of Florence, Italy

Languages

English

read, spoken and written (incl. scientific English)

French

Native

IT skills

JAVA - PYTHON

Experiences

Jan 2016 - June 2018

Project manager for the development of the « Deep Blue Process » start-up project.

- Methodological development for the maturation of the Deep Blue Process technology .

- Business development for the futur Deep Blue Process start-up.

Jan 2015 - June 2016

Postdoc in the Extremophiles and Large Molecular Assemblies group headed by the Dr. Bruno Franzetti at the « Institut de Biologie Structurale », Grenoble, France:

- Methodological development for the characterization of new enzymes activities by HPLC.

- Proof of concept development for the dissemination and exploitation of R&D results.

2010 - 2014

Young researcher (in PhD) in the Extremophiles and Large Molecular Assemblies group headed by the Dr. Bruno Franzetti at the « Institut de Biologie Structurale », Grenoble, France : Deciphering the role and the mode of action of a family of ATP-independent aminopeptidases in hyperthermophilic Archaea.

- Expression, purification, biochemical (enzymatic, activity, stability) and biophysical (structure, mode of assembling) characterization of TET proteins.

- Structural and phylogenetic analysis in collaboration with S.Gribaldo at the Pasteur Institut, Paris

1st Semester 2010

Master 2 Internship at the Institut Laue Langevin in the Large Scale Structure Group headed by the Pr. Joseph Zaccai :

- Cloning, expression and purification of a mutated hyperthermophilic protein from Pyrococcus horikoshii and stabilizing conditions adjustment, study of the low resolution envelope of the protein by Small Angle X-ray/Neutron Scattering.

Feb - May 2009

Master 1 intership at the Pasteur Institut in Paris, France, in the Yeasts Molecular Genetics Unit, headed by Pr. Bernard Dujon: study of the spreading of trinucleotide repetition in S. cerevisiae.

-Transformation of S.cerevisiae yeast, yeast culture in tube and in 96-well plate, purification of yeast DNA S.cerevisiae in 96-well plate, study of the DNA repetition by gel electrophoresis and radioactive labeling.

Interests

  • Archery
  • Photography
    digital & analog
    development
  • Guitar

alexandre.appolwall-eaire@gmail.com

ALEXANDRE APPOLAIRE
Intitut de Biologie Structurale
Campus EPN
71 avenue des Martyrs
CS 10090
38044 GRENOBLE CEDEX 9
PHONE: 00.33.(0)4.57.425852.87.93

Merci.
Votre message a bien été envoyé.

Please fill the mandatory fields
A technical error has occured

* Mandatory fields