80 research outputs found
Heterologous expression of three enzymes forstructural studies
In questo lavoro di tesi ci si \ue8 occupati dell\u2019espressione, della purificazione e della
cristallizzazione di tre enzimi (bile acid CoA:amino acid N-acyltransferase,
BAAT; farnesyl cysteine-carboxyl methyltransferase, Ste14; e stearoyl-CoA
desaturase, SCD) con lo scopo finale di determinarne la struttura tridimensionale
mediante analisi di diffrazione di raggi X.
I cDNA delle proteine in questione sono stati clonati in vettori per l\u2019espressione in
sistemi eterologhi. Le scelta del sistema di espressione opportuno per la
produzione su larga scala \ue8 stata condotta dopo una valutazione della resa, della
stabilit\ue0 ed integrit\ue0 del prodotto proteico.
La proteina BAAT umana \ue8 stata espressa utilizzando il sistema procariotico
E.coli. La proteina ricombinante \ue8 stata purificata tramite due cromatografie di
affinit\ue0 al nichel e gel filtrazione. L\u2019integrit\ue0 del campione ottenuto \ue8 stata
controllata attraverso spettrometria di massa e l\u2019omogeneit\ue0 mediante DLS. E\u2019
stata condotta anche una valutazione dell\u2019attivit\ue0 enzimatica verificando la
presenza dei prodotti mediante spettrometria di massa. Oltre alla proteina wild
type \ue8 stato prodotto il mutante privo di attivit\ue0 enzimatica (C235A) per procedere
con le prove di cristallizzazione in presenza del substrato. I campioni ottenuti si
presentano sufficientemente puri, stabili ed omogenei per allestire le prove di
cristallizzazione. Le prove di cristallizzazione hanno dato esito positivo con la
proteina mutata, anche se i cristalli fino ad ora ottenuti non sono idonei per gli
esperimenti di diffrazione di raggi X.
La proteina di membrana Ste14 di C.glabrata \ue8 stata individuata come buon
candidato per la produzione su larga scala al fine di condurre studi strutturali
attraverso l\u2019utilizzo di un sistema high-throughput pubblicato da Drew et al.,
2008. Il sistema prevede l\u2019espressione eterologa in S. cerevisiae; la proteina di
interesse viene prodotta in fusione alla GFP permettendo la valutazione del livello
di espressione e la stabilit\ue0 nei diversi detergenti attraverso misure di fluorescenza
sull\u2019estratto cellulare. L\u2019enzima \ue8 stato purificato attraverso due cromatografie di
affinit\ue0 al nichel e gel filtrazione in presenza sia del detergente DDM sia del
detergente LDAO. Il profilo della gel filtrazione suggerisce che il campione \ue8
\u2018monodisperso\u2019 e quindi adeguato per l\u2019allestimento di prove di cristallizzazione.
Ad oggi non sono per\uf2 stati ottenuti cristalli adatti ad esperimenti di diffrazione.
E\u2019 stato infine messo appunto un protocollo di espressione e purificazione per
l\u2019enzima transmembrana umano SCD. La proteina ricombinante \ue8 stata espressa
in cellule di insetto e purificata in presenza del detergente DDM attraverso IMAC.
La resa e la purezza del prodotto proteico ad oggi non \ue8 ancora ottimale ma la
quantit\ue0 di enzima \ue8 sufficiente per allestire prove di cristallizzazione e per
condurre studi biochimici e strutturali.This thesis work was aimed at the expression, purification and crystallization of
three human proteins (bile acid CoA:amino acid N-acyltransferase, BAAT;
farnesyl cysteine-carboxyl methyltransferase, Ste14; and stearoyl-CoA desaturase,
SCD) in order to determine their three-dimensional structure using X-ray
crystallography.
The cDNA sequences were cloned into specific vectors for overexpression in
heterologous systems. The choice of the best expression system was made
considering the protein yield, stability and integrity before scaling up.
Human BAAT was expressed in E.coli. The recombinant protein was purified by
IMAC, reverse IMAC and gel filtration. The integrity of the sample was assessed
by mass spectrometry and its homogeneity using DLS. The enzymatic activity
was controlled verifying the presence of the products by mass spectrometry. In
addition to the wild type protein, the catalytic mutant (C235A) was cloned and
expressed to carry out crystallization trials in the presence of the substrate. The
samples obtained are sufficiently pure, stable and homogeneous to set up
crystallization trials. Crystals were obtained only with the mutant but they are not
sufficiently ordered for X-ray diffraction experiments.
Using the high-throughput system published by Drew et al., 2008, the membrane
protein Ste14 from C.glabrata was selected as a good candidate for a large scale
production in order to conduct structural studies. The system uses S.cerevisiae to
overexpress membrane proteins; the protein of interest is cloned into a GFPfusion
vector allowing to estimate the expression level and the stability in several
detergents by measuring fluorescence directly in cell extracts. The enzyme was
purified by IMAC, reverse IMAC and gel filtration in the presence of the
detergents DDM and LDAO. The gel filtration profile indicates that the sample is
\u2018monodisperse\u2019 and hence adequate to set crystallization trials. Up to now no
suitable crystals were obtained.
Finally, a protocol for the expression and purification of the human membrane
enzyme SCD was developed. The recombinant protein was expressed in insect
cells and purified in the presence of the detergent DDM by IMAC. Until now the
yield and the purity obtained are not optimal but a sufficient quantity of the
enzyme can be extracted to carry out crystallization trials aimed at structural and
biochemical studies
Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation
Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies
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Genetic, structural, and molecular insights into the function of ras of complex proteins domains
Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored
Leucine-rich repeat kinase 2 and alpha-synuclein: intersecting pathways in the pathogenesis of Parkinson's disease?
Although Parkinson's disease (PD) is generally a sporadic neurological disorder, the discovery of monogenic, hereditable forms of the disease has been crucial in delineating the molecular pathways that lead to this pathology. Genes responsible for familial PD can be ascribed to two categories based both on their mode of inheritance and their suggested biological function. Mutations in parkin, PINK1 and DJ-1 cause of recessive Parkinsonism, with a variable pathology often lacking the characteristic Lewy bodies (LBs) in the surviving neurons. Intriguingly, recent findings highlight a converging role of all these genes in mitochondria function, suggesting a common molecular pathway for recessive Parkinsonism. Mutations in a second group of genes, encoding alpha-synuclein (α-syn) and LRRK2, are transmitted in a dominant fashion and generally lead to LB pathology, with α-syn being the major component of these proteinaceous aggregates. In experimental systems, overexpression of mutant proteins is toxic, as predicted for dominant mutations, but the normal function of both proteins is still elusive. The fact that α-syn is heavily phosphorylated in LBs and that LRRK2 is a protein kinase, suggests that a link, not necessarily direct, exists between the two. What are the experimental data supporting a common molecular pathway for dominant PD genes? Do α-syn and LRRK2 target common molecules? Does LRRK2 act upstream of α-syn? In this review we will try to address these of questions based on the recent findings available in the literature
Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation
Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field
GTPase activity regulates kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2
Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneratio
LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate
Background
Lrrk2, a gene linked to Parkinson\u2019s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins.
Results
Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity.
Conclusions
Given that the most common Parkinson\u2019s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF
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PAK6 phosphorylates 14-3-3γ to regulate steady state phosphorylation of LRRK2
Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain
The Roc domain of LRRK2 as a hub for protein-protein interactions:a focus on PAK6 and its impact on RAB phosphorylation
Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell
PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson’s disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2’s phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation
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