A metamorfose das necessidades em desejos / The metamorphosis of the needs in desires

Lacan e Kojève "modernizaram" a ideia hegeliana do desejo colocado como essência da espécie humana, inspirando-se na linguística estruturalista que inscreve o sujeito na linguagem. O capitalismo determina social e historicamente esse desejo, já que as trocas mercantis constituem uma linguagem grosseira, a "linguagem que as mercadorias falam entre si", segundo a feliz frase de Marx. Disso resulta que o propósito aparentemente racional das compras, o imperativo de satisfazer as necessidades vitais finitas, é metamorfoseado na busca compulsiva e inconsciente por desejos venais inacessíveis e infinitos - compulsão assimilável à perversão sexual do fetichismo - o que abre perspectivas de mercantilização ilimitada à globalização capitalista.(AU)

Lacan and Kojève have "modernized" the Hegelian conception of desire placed as the essence of the mankind, based on structuralist linguistics that inscribes the subject in language. Capitalism determines socially and historically this desire because the mercantile exchanges are a rough language, "the language that the goods speak to each other", in Marx's happy expression. As a result, the seemingly rational purpose of purchases, the need to meet finite vital needs, is morphed into compulsive and unconscious search for inaccessible and infinite venal desires - similar to compulsion in the sexual perversion of fetishism - which opens prospects for unlimited commercialization for the capitalist globalization.(AU)

A metamorfose das necessidades em desejos / The metamorphosis of the needs in desires

Lacan e Kojève "modernizaram" a ideia hegeliana do desejo colocado como essência da espécie humana, inspirando-se na linguística estruturalista que inscreve o sujeito na linguagem. O capitalismo determina social e historicamente esse desejo, já que as trocas mercantis constituem uma linguagem grosseira, a "linguagem que as mercadorias falam entre si", segundo a feliz frase de Marx. Disso resulta que o propósito aparentemente racional das compras, o imperativo de satisfazer as necessidades vitais finitas, é metamorfoseado na busca compulsiva e inconsciente por desejos venais inacessíveis e infinitos - compulsão assimilável à perversão sexual do fetichismo - o que abre perspectivas de mercantilização ilimitada à globalização capitalista.

Lacan and Kojève have "modernized" the Hegelian conception of desire placed as the essence of the mankind, based on structuralist linguistics that inscribes the subject in language. Capitalism determines socially and historically this desire because the mercantile exchanges are a rough language, "the language that the goods speak to each other", in Marx's happy expression. As a result, the seemingly rational purpose of purchases, the need to meet finite vital needs, is morphed into compulsive and unconscious search for inaccessible and infinite venal desires - similar to compulsion in the sexual perversion of fetishism - which opens prospects for unlimited commercialization for the capitalist globalization.

SAXS and X-ray crystallography suggest an unfolding model for the GDP/GTP conformational switch of the small GTPase Arf6.

The small GTPases Arf1 and Arf6 have nonoverlapping functions in cellular traffic despite their very high sequence and structural resemblance. Notably, the exquisite isoform specificity of their guanine nucleotide exchange factors and their distinctive sensitivity to the drug brefeldin A cannot be explained by any straightforward structural model. Here we integrated structural and spectroscopic methods to address this issue using Δ13Arf6-GDP, a truncated mutant that mimics membrane-bound Arf6-GDP. The crystal structure of Δ13Arf6-GDP reveals an unprecedented unfolding of the GTPase core ß-strands, which is fully accounted for by small-angle X-ray scattering data in solution and by ab initio three-dimensional envelope calculation. NMR chemical shifts identify this structural disorder in Δ13Arf6-GDP, but not in the closely related Δ17Arf1-GDP, which is consistent with their comparative thermodynamic and hydrodynamic analyses. Taken together, these experiments suggest an unfolding model for the nucleotide switch of Arf6 and shed new light on its biochemical differences with Arf1.

An Arf1 GTPase mutant with different responses to GEF inhibitors.

Guanine nucleotide exchange factors (GEFs) stimulate the activation of small GTP-binding proteins (GTPases). Establishing their specificity is a challenging issue, in which chemical genetics are rapidly gaining interest. We report a mutation in the Arf1 GTPase, K38A, which differentially alters its sensitivity to GEF inhibitors. The mutation renders Arf1 insensitive to LM11, a GEF inhibitor that we previously discovered by structure-based screening. In contrast, full inhibition by the natural compound Brefeldin A (BFA) is retained. We show that the mutation is otherwise silent towards the biochemical and cellular properties of Arf1, notably its binding to effectors as measured by a novel GEF-protection assay. This is thus the first GTPase mutant with different responses to two classes of inhibitors, and a novel tool to analyze Arf and ArfGEF specificity and functions in vitro and in cells.

Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.

Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.

Dual specificity of the interfacial inhibitor brefeldin a for arf proteins and sec7 domains.

Guanine nucleotide exchange factors (GEFs), which activate small GTP-binding proteins (SMG) by stimulating their GDP/GTP exchange, are emerging as candidate targets for the inhibition of cellular pathways involved in diseases. However, their specific inhibition by competitive inhibitors is challenging, because GEF and SMG families comprise highly similar members. Nature shows us an alternative strategy called interfacial inhibition, exemplified by Brefeldin A (BFA). BFA inhibits the activation of Arf1 by its GEFs in vivo by stabilizing an abortive complex between Arf-GDP and the catalytic Sec7 domain of some of its GEFs. Here we characterize the specificity of BFA toward wild-type (ARNO and BIG1) and mutant Sec7 domains and toward class I, II, and III Arfs. We find that BFA sensitivity of the exchange reaction depends on the nature of both the Sec7 domain and the Arf protein. A single Phe/Tyr substitution is sufficient to achieve BFA sensitivity of the Sec7 domain, which is supported by our characterization of brefeldin C (BFC), a BFA analog that cannot interact with the Tyr residue, and by free energy computations. We further show that Arf1 and Arf5, but not Arf6, are BFA-sensitive, despite their having every BFA-interacting residue in common. Analysis of Arf6 mutants points to the dynamics of the interswitch, which is involved in membrane-to-nucleotide signal propagation, as contributing to, although not sufficient for, BFA sensitivity. Altogether, our results reveal the Tyr/Phe substitution as a novel tool for monitoring BFA sensitivity of cellular ArfGEFs and document the exquisite and dual specificity that can be achieved by an interfacial inhibitor.

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor.

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.

Transforming growth factor beta1 overexpression in the nigrostriatal system increases the dopaminergic deficit of MPTP mice.

Idiopathic Parkinson's disease (PD) is characterized by mesencephalic dopaminergic neuron cell death and striatal dopamine (DA) depletion. The factors involved in the pathogenesis of the disease are still unknown. Transforming growth factor beta1 (TGFbeta1) is increased in the striatum of patients with PD. However, the effect of this increase is not known. Here, we show that overexpression of TGFbeta1, by recombinant adenovirus TGFbeta1 gene transfer, in the mesostriatal system of an MPTP mouse model of PD decreased the number of mesencephalic dopaminergic neurons. This effect also involved more extensive DA depletion in the striatum. Striatal enkephalin mRNA levels are augmented, suggesting a decrease in dopaminergic transmission to the postsynaptic target. In the absence of MPTP, TGFbeta1 greatly decreased the number of dopaminergic neurons in the ventral mesencephalon of fully mature mice. These results show that an increase in TGFbeta1 levels aggravate the parkinsonian status of MPTP mice and may therefore be a risk factor for the development of PD.

Mechanism of domain closure of Sec7 domains and role in BFA sensitivity.

Activation of small G proteins of the Arf family is initiated by guanine nucleotide exchange factors whose catalytic Sec7 domain stimulates the dissociation of the tightly bound GDP nucleotide. The exchange reaction involves distinct sequential steps that can be trapped by the noncompetitive inhibitor brefeldin A, by mutation of an invariant catalytic glutamate, or by removal of guanine nucleotides. Arf-GDP retains most characteristics of its GDP-bound form at the initial low-affinity Arf-GDP-Sec7 step. It then undergoes large conformational changes toward its GTP-bound form at the next step, and eventually dissociates GDP to form a nucleotide-free high-affinity Arf-Sec7 complex at the last step. Thus, Arf proteins evolve through different conformations that must be accommodated by Sec7 domains in the course of the reaction. Here the contribution of the flexibility of Sec7 domains to the exchange reaction was investigated with the crystal structure of the unbound Sec7 domain of yeast Gea2. Comparison with Gea2 in complex with nucleotide-free Arf1 Delta 17 [Goldberg, J. (1998) Cell 95, 237-248] reveals that Arf induces closure of the two subdomains that form the sides of its active site. Several residues that determine sensitivity to brefeldin A are involved in interdomain and local movements, pointing to the importance of the flexibility of Sec7 domains for the inhibition mechanism. Altogether, this suggests a model for the initial steps of the exchange reaction where Arf docks onto the C-terminal domain of the Sec7 domain before closure of the N-terminal domain positions the catalytic glutamate to complete the reaction.