P-Rex1 is a novel substrate of the E3 ubiquitin ligase Malin associated with Lafora disease.

Laforin and Malin are two proteins that are encoded by the genes EPM2A and EPM2B, respectively. Laforin is a glucan phosphatase and Malin is an E3-ubiquitin ligase, and these two proteins function as a complex. Mutations occurring at the level of one of the two genes lead to the accumulation of an aberrant form of glycogen meant to cluster in polyglucosans that go under the name of Lafora bodies. Individuals affected by the appearance of these polyglucosans, especially at the cerebral level, experience progressive neurodegeneration and several episodes of epilepsy leading to the manifestation of a fatal form of a rare disease called Lafora disease (LD), for which, to date, no treatment is available. Despite the different dysfunctions described for this disease, many molecular aspects still demand elucidation. An effective way to unknot some of the nodes that prevent the achievement of better knowledge of LD is to focus on the substrates that are ubiquitinated by the E3-ubiquitin ligase Malin. Some substrates have already been provided by previous studies based on protein-protein interaction techniques and have been associated with some alterations that mark the disease. In this work, we have used an unbiased alternative approach based on the activity of Malin as an E3-ubiquitin ligase. We report the discovery of novel bonafide substrates of Malin and have characterized one of them more deeply, namely PIP3-dependent Rac exchanger 1 (P-Rex1). The analysis conducted upon this substrate sets the genesis of the delineation of a molecular pathway that leads to altered glucose uptake, which could be one of the origin of the accumulation of the polyglucosans present in the disease.

Solution structure of a protein denatured state and folding intermediate.

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation.

The Engrailed Homeodomain protein has the highest refolding and unfolding rate constants directly observed to date. Temperature jump relaxation measurements gave a refolding rate constant of 37,500 s(-1) in water at 25 degrees C, rising to 51,000 s(-1) around 42 degrees C. The unfolding rate constant was 1,100 s(-1) in water at 25 degrees C and 205,000 s(-1) at 63 degrees C. The unfolding half-life is extrapolated to be approximately 7.5 ns at 100 degrees C, which allows real-time molecular dynamics unfolding simulations to be tested on this system at a realistic temperature. Preliminary simulations did indeed conform to unfolding on this time scale. Further, similar transition states were observed in simulations at 100 degrees C and 225 degrees C, suggesting that high-temperature simulations provide results applicable to lower temperatures.

From snapshot to movie: phi analysis of protein folding transition states taken one step further.

Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by phi analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation-condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.