Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant.

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.

Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.

Luminescence, absorption, and Stern-Volmer studies of cerium chloride and nitrate compounds in acidic and neutral aqueous, and non-aqueous solutions.

Complexation of cerium chloride and nitrate in neutral and acidic aqueous solutions as well as in anhydrous alcohol solutions were investigated using emission, excitation, and absorption spectroscopic techniques. In aqueous solution cerium chloride shows a strong, and broad emission centering at 365 nm. The excitation spectra are observed at 266 and 296 nm with the shorter wavelength showing the highest intensity. Cerium chloride compound also strongly emits in methanol (MeOH), where the broad emission spectrum is red shifted by ∼10-375 nm. The excitation spectrum in MeOH shows bands at 255 and 309 nm, respectively with the longer wavelength band (at 309 nm) dominating. The relative intensities of these two excitation bands are reversed in protic aqueous solution. In contrast, solutions of cerium nitrate are only weakly luminescent in aqueous media, while the emission is totally quenched in MeOH solution. These observations indicate that the spectral profiles are largely influenced by the extent of inner-sphere coordination and the type of the dominant species in solutions. Both nitrate and chloride anions show enhanced inner-sphere coordination in MeOH when compared with that of the aqueous media. However, enhanced inner-sphere complexation of the NO(3)(-) ion quenches the emission, while the reverse effect is observed upon Cl(-) coordination. Stern-Volmer studies provide quenching constant, K(sv), value of 577M(-1). The calculated rate constant k(r) is 1.3×10(10)M(-1)s(-1) indicating diffusion controlled bimolecular process as the major mode of interaction.

Influence of posture and stimulus parameters on post-activation depression of the soleus H-reflex in individuals with chronic spinal cord injury.

In non-disabled (ND) individuals, reflexes are modulated by influences related to physiologic state (e.g., posture, joint position, load) and activation history. Repeated activation of the H-reflex results in post-activation depression (PAD) of the response amplitude. The modulation associated with physiologic state and activation history is suppressed or abolished in individuals with spinal cord injury (SCI). While posture is known to affect H-reflex amplitude and PAD in non-disabled individuals, the effect of posture on PAD in SCI individuals is not known. Further, while the amount of PAD is also known to be influenced by the stimulus rate and by the amplitude of the evoked reflex, the interaction of posture with stimulus parameters has not been previously investigated in either group. We investigated differences in PAD of the soleus H-reflex between SCI subjects and ND subjects during sitting versus supported standing. Subjects were tested using paired conditioning-test stimulus pulses of 2.5s and 5s interpulse intervals (ISI) and with stimulus intensity adjusted to evoke reflex responses of 20% and 40% of the maximum motor response. We found standing posture to be associated with significantly less PAD in SCI subjects compared to ND subjects. In both groups, shorter ISIs and smaller reflex amplitudes were associated with greater PAD of the H-reflex. These results indicate that postural influences on post-activation modulation, while present, are impaired in individuals with chronic incomplete SCI.

Rapid functional recovery after spinal cord injury in young rats.

Responses to traumatic injury in the immature spinal cord may be different from those in adults. We modified an adult model of weight-drop injury to characterize the histopathology and functional recovery after spinal cord injury (SCI) in rat pups at postnatal day 14-15. A 10-g weight was dropped from 2.5 or 5.0 cm at T8-T9. Hindlimb function was evaluated at 24 h and 1, 2, 3, and 4 weeks after injury using the Combined Behavioral Score that estimates overall hind limb sensorimotor function, and the BBB scale for open field locomotion. Histopathology was examined at 15 min, 24 h, and 4 weeks after SCI. The initial hemorrhagic lesion was similar to that seen in adults, but the time course of secondary loss of ventral horn motor neurons was extended. By 4 weeks, only a partial rim of white matter surrounding a central cavity was seen. The 5.0 cm injury group exhibited significantly less recovery of function at 4 weeks than the 2.5 cm group. In the latter, the degree of hindlimb deficit at 4 weeks was similar to that previously described for adults with 10 g x 2.5 cm SCI. However, pups in both injury groups exhibited a significantly faster rate of recovery than adults. Recovery was maximal by 1 week after SCI in pups as compared to 3-4 weeks in adults. The more rapid functional recovery observed in the pups suggests that this new model may be useful for studying mechanisms of functional plasticity after SCI.

Glutamate receptor subunit expression after spinal cord injury in young rats.

To investigate the possibility that glutamate receptor levels in the spinal cord are altered following injury to young rats, we used a previously characterized model of spinal cord contusion that produces a reliable injury in rats at postnatal day 14-15. Quantitative Western blot analysis was used to measure relative amounts of protein for several glutamate receptor subunits acutely (24 h) and chronically (28 days) after spinal cord injury (SCI). Acutely after injury significant decreases were observed in the GluR1, GluR2, and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor, and the NR2A and NR2B subunits, but not the NR1 subunit, of the N-methyl-d-aspartate (NMDA) receptor. However, 28 days after injury only one subunit (GluR4) was shown to be altered. These widespread changes that occur acutely in receptor subunit expression may be an attempt to protect cells from glutamate-induced death. The injured spinal cord in these young animals, however, appears to have the capacity to regulate receptor subunit levels to normal within a month of injury.

Quantitative measurement of glutamate receptor subunit protein expression in the postnatal rat spinal cord.

Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits (N-methyl-D-aspartate (NMDA) subunits NR1, NR2A, NR2B, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.