[Plasmid pJP4 mediated gene-augmentation in different systems and its effect on 2,4-D biodegradation].

With pJP4 plasmid carrying genetic engineering bacteria Pseudomonas putida SM1443 :: gfp2x (pJP4 :: dsRed) as the donor, horizontal gene transfer of pJP4 plasmid in 4 isolated pure strains was investigated, and effects of the donor bacteria inoculation on the removal of the target pollutant 2,4-dichlorophenoxyacetic acid (2,4-D) was studied through conducting gene augmentation in activated sludge, biofilm, aerobic granular sludge and river sediment system, respectively. Results showed that plasmid pJP4 could transfer from Pseudomonas putida SM1443 to a broad spectrum of bacteria. Inoculation of pJP4 plasmid carrying donor bacterium apparently promoted the degradation of 2,4-D for all the above four systems. For the activated sludge system (2,4-D initial concentration at 450 mg/L), 66% and 54% removal of 2,4-D was achieved after 143.5 h reaction for the gene augmented and control system, respectively. For the biofilm system with 2,4-D initial concentration at 180 mg/L, 2,4-D removal percentage at 113 h was 99% and 61%, respectively. For aerobic granular sludge system (2,4-D initial concentration at 160 mg/L), 2,4-D was nearly completely removed by 62 h in the gene-augmented system, while the control system only degraded 26% at 66 h. For the system with sediment (2,4-D initial concentration at 2 mg/L), 93% and 69% removal of 2,4-D was obtained at 344 h reaction for the gene augmented and control system, respectively. Confocal laser scanning microscopy (CLSM) analysis revealed the formation and presence of transconjugants in different gene augmentation systems.

Ubiquitin C-terminal hydrolase-L3-knockout mice are resistant to diet-induced obesity and show increased activation of AMP-activated protein kinase in skeletal muscle.

Obesity results from the dysregulation of energy balance throughout the entire body. Although the ubiquitin system participates in many cellular processes, its contribution to the balance of energy in the body remains poorly understood. Here, we show that ubiquitin C-terminal hydrolase (UCH)-L3, one of the deubiquitinating enzymes, contributes to the regulation of metabolism. Uchl3(-/-) mice displayed a reduction of adipose tissue mass and were protected against high-fat diet (HFD)-induced obesity and insulin resistance. Uchl3(-/-) mice given both a normal chow and an HFD had an increased whole-body energy expenditure accounting for the reduction of adipose tissue mass. Activation of AMP-activated protein kinase (AMPK) in skeletal muscle has been reported to increase fatty acid beta-oxidation, leading to the elevation of the whole-body energy expenditure. Consistently, increased activation of AMPK and fatty acid beta-oxidation was observed in skeletal muscle of Uchl3(-/-) mice. Mouse embryonic fibroblasts derived from Uchl3(-/-) mice also showed increased activation of AMPK, indicating that UCH-L3 is involved in a cell-autonomous down-regulation of AMPK. These results suggest a role for UCH-L3 in the regulation of AMPK activity and whole-body energy metabolism.

Proteomic and histochemical analysis of proteins involved in the dying-back-type of axonal degeneration in the gracile axonal dystrophy (gad) mouse.

Local axonal degeneration is a common pathological feature of peripheral neuropathies and neurodegenerative disorders of the central nervous system, including Alzheimer's disease, Parkinson's disease, and stroke; however, the underlying molecular mechanism is not known. Here, we analyzed the gracile axonal dystrophy (gad) mouse, which displays the dying-back-type of axonal degeneration in sensory neurons, to find the molecules involved in the mechanism of axonal degeneration. The gad mouse is analogous to a null mutant of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). UCH-L1 is a deubiquitinating enzyme expressed at high levels in neurons, as well as testis and ovary. In addition, we recently discovered a new function of UCH-L1-namely to bind to and stabilize mono-ubiquitin in neurons, and found that the level of mono-ubiquitin was decreased in neurons, especially in axons of the sciatic nerve, in gad mice. The low level of ubiquitin suggests that the target proteins of the ubiquitin proteasome system are not sufficiently ubiquitinated and thus degraded in the gad mouse; therefore, these proteins may be the key molecules involved in axonal degeneration. To identify molecules involved in axonal degeneration in gad mice, we compared protein expression in sciatic nerves between gad and wild-type mice at 2 and 12 weeks old, using two-dimensional difference gel electrophoresis. As a result, we found age-dependent accumulation of several proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 14-3-3, in gad mice compared with wild-type mice. Histochemical analyses demonstrated that GAPDH and 14-3-3 were localized throughout axons in both gad and wild-type mice, but GAPDH accumulated in the axons of gad mice. Recently, it has been suggested that a wide range of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates, and it has been reported that oxidative stress causes the aggregation of GAPDH. Furthermore, histochemical analysis demonstrated that sulfonated GAPDH, a sensor of oxidative stress that elicits cellular dysfunction, was expressed in the axons of gad mice, and 4-hydroxy-2-nonenal, a major marker of oxidative stress, was also only detected in gad mice. Our findings suggest that GAPDH may participate in a process of the dying-back-type of axonal degeneration in gad mice and may provide valuable insight into the mechanisms of axonal degeneration.

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant.

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Photoreceptor cell apoptosis in the retinal degeneration of Uchl3-deficient mice.

UCH-L3 belongs to the ubiquitin C-terminal hydrolase family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. A murine Uchl3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. To elucidate the function of UCH-L3, we investigated histopathological changes and expression of apoptosis- and oxidative stress-related proteins during retinal degeneration. In the normal retina, UCH-L3 was enriched in the photoreceptor inner segment that contains abundant mitochondria. Although the retina of Uchl3-deficient mice showed no significant morphological abnormalities during retinal development, prominent retinal degeneration became manifested after 3 weeks of age associated with photoreceptor cell apoptosis. Ultrastructurally, a decreased area of mitochondrial cristae and vacuolar changes were observed in the degenerated inner segment. Increased immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment indicated mitochondrial oxidative stress. Expression of cytochrome c, caspase-1, and cleaved caspase-3 did not differ between wild-type and mutant mice; however, immunoreactivity for endonuclease G was found in the photoreceptor nuclei in the mutant retina. Hence, loss of UCH-L3 leads to mitochondrial oxidative stress-related photoreceptor cell apoptosis in a caspase-independent manner. Thus, Uchl3-deficient mice represent a model for adult-onset retinal degeneration associated with mitochondrial impairment.

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice.

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.

Clinico-pathological rescue of a model mouse of Huntington's disease by siRNA.

Huntington's disease (HD) is an autosomal dominant inheritable neurodegenerative disorder currently without effective treatment. It is caused by an expanded polyglutamine (poly Q) tract in the corresponding protein, huntingtin (htt), and therefore suppressing the huntingtin expression in brain neurons is expected to delay the onset and mitigate the severity of the disease. Here, we have used small interfering RNAs (siRNAs) directed against the huntingtin gene to repress the transgenic mutant huntingtin expression in an HD mouse model, R6/2. Results showed that intraventricular injection of siRNAs at an early postnatal period inhibited transgenic huntingtin expression in brain neurons and induced a decrease in the numbers and sizes of intranuclear inclusions in striatal neurons. Treatments using this siRNA significantly prolonged model mice longevity, improved motor function and slowed down the loss of body weight. This work suggests that siRNA-based therapy is promising as a future treatment for HD.

[Clinical trial and evaluation on comprehensive treatment on attack in acute stage: report of 522 cases].

OBJECTIVE: To investigate the clinical effect of the comprehensive treatment to acute stage of attack. METHODS: On the basis of the previous observation, the study of the randomization control with general treatment, treatment on acupuncture and western medical treatment were carried out. A comprehensive treatment on overall traditional Chinese medical differentiation according to the superiority of every treatment was assessed and evaluated in 522 patients with attack. RESULTS: The comprehensive treatment of cerebral infarction was superior to the western medicine treatment. General treatment, treatment on traditional Chinese medical differentiation, acupuncture group revealed different improvement on neural function, daily viability, cognitive function in various extent. CONCLUSION: The comprehensive treatment that based on overall traditional Chinese medical differentiation has advantage and characteristic. It has positive combined action to the attack and relevant to clinical setting, easier to popularization and application. Various appraising amount form has different evaluating effects in different stage.

Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis.

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.

Potentiation of ATP-induced currents due to the activation of P2X receptors by ubiquitin carboxy-terminal hydrolase L1.

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity.

Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.

The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis.

Accumulation of beta- and gamma-synucleins in the ubiquitin carboxyl-terminal hydrolase L1-deficient gad mouse.

The synuclein family includes three isoforms, termed alpha, beta and gamma. alpha-Synuclein accumulates in various pathological lesions resulting from neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. However, neither beta- nor gamma-synuclein has been detected in Lewy bodies, and thus it is unclear whether these isoforms contribute to neurological pathology. In the present study, we used immunohistochemistry to demonstrate accelerated accumulation of beta- and gamma-synucleins in axonal spheroids in gracile axonal dystrophy (gad) mice, which do not express ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). gamma-Synuclein immunoreactivity in the spheroids appeared in the gracile nucleus at 3 weeks of age and was maintained until 32 weeks. beta-Synuclein immunoreactivity appeared in spheroids around 12 weeks of age. In contrast, alpha-synuclein immunoreactivity was barely detectable in spheroids. Immunoreactivity for synaptophysin and ubiquitin were either faint or undetectable in spheroids. Given that UCH-L1 deficiency results in axonal degeneration and spheroid formation, our findings suggest that beta- and gamma-synuclein participate in the pathogenesis of axonal swelling in gad mice.

Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.

The ubiquitin pathway functions in the process of protein turnover in eukaryotic cells. This pathway comprises the enzymes that ubiquitinate/deubiquitinate target proteins and the proteasome that degrades ubiquitin-conjugated proteins. Ubiquitin C-terminal hydrolases (UCHs) are thought to be essential for maintaining ubiquitination activity by releasing ubiquitin (Ub) from its substrates. Mammalian UCH-L1 and UCH-L3 are small proteins that share considerable homology at the amino acid level. Both of these UCHs are highly expressed in the testis/ ovary and neuronal cells. Our previous work demonstrated that UCH-L1-deficient gracile axonal dystrophy (gad) mice exhibit progressively decreasing spermatogonial stem cell proliferation, suggesting that UCH isozymes in the testis function during spermatogenesis. To analyze the expression patterns of UCH isozymes during spermatogenesis, we isolated nearly homogeneous populations of spermatogonia, spermatocytes, spermatids, and Sertoli cells from mouse testes. Western blot analysis detected UCH-L1 in spermatogonia and Sertoli cells, whereas UCH-L3 was detected in spermatocytes and spermatids. Moreover, reverse transcription-polymerase chain reaction analysis of UCH isozymes showed that UCH-L1 and UCH-L4 mRNAs are expressed in spermatogonia, whereas UCH-L3 and UCH-L5 mRNAs are expressed mainly in spermatocytes and spermatids. These results suggest that UCH-L1 and UCH-L3 have distinct functions during spermatogenesis, namely, that UCH-L1 may act during mitotic proliferation of spermatogonial stem cells whereas UCH-L3 may function in the meiotic differentiation of spermatocytes into spermatids.

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins.

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.

Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed approximately 70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.

Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.