Expression of Human L-Dopa Decarboxylase (DDC) under Conditions of Oxidative Stress.

Oxidative stress is known to influence mRNA levels, translation, and proteolysis. The importance of oxidative stress has been demonstrated in several human diseases, including neurodegenerative disorders. L-Dopa decarboxylase (DDC) is the enzyme that converts L-Dopa to dopamine (DA). In spite of a large number of studies, little is known about the biological significance of the enzyme under physiological and pathological conditions. Here, we investigated the relationship between DDC expression and oxidative stress in human neural and non-neural cells. Oxidative stress was induced by treatment with H2O2. Our data indicated that mRNA and protein expression of DDC was enhanced or remained stable under conditions of ROS induction, despite degradation of total RNA and increased cytotoxicity and apoptosis. Moreover, DDC silencing caused an increase in the H2O2-induced cytotoxicity. The current study suggests that DDC is involved in the mechanisms of oxidative stress.

L-Dopa-Decarboxylase (DDC) Is a Positive Prognosticator for Breast Cancer Patients and Epinephrine Regulates Breast Cancer Cell (MCF7 and T47D) Growth In Vitro According to Their Different Expression of Gi- Protein- Coupled Receptors.

A coherence between thyroid dysfunction and breast cancer incidence exists. Thyroid hormone metabolites bind to TAAR1 (trace amine-associated receptor 1) and through that modulate the serotonergic and dopaminergic system. Catecholamines themselves are synthesized by the L-dopa decarboxylase (DDC). The aim of our study was to analyze the influence of catecholamines on the DDC expression in primary breast cancer patients and the role of DDC concerning overall survival (OS). DDC expression was analyzed by immunohistochemistry. The effect of epinephrine on the expression of DDC and the Gi- protein was analyzed on the protein level via Western blot. A viability assay was performed to test the metabolic cell viability. The overexpression of DDC in the primary tumor was associated with longer OS (p = 0.03). Stimulation with epinephrine induced the downregulation of DDC (p = 0.038) and significantly increased viability in T47D cells (p = 0.028). In contrast, epinephrine induced an upregulation of DDC and decreased the proliferation of MCF7 cells (p = 0.028). Epinephrine led to an upregulation of Gi protein expression in MCF7 cells (p = 0.008). DDC is a positive prognostic factor for OS in breast cancer patients, and it is regulated through epinephrine differently in MCF7 and T47D. DDC may represent a novel target for the treatment of breast cancer, especially concerning its interaction with epinephrine.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

Significance of Catecholamine Biosynthetic/Metabolic Pathway in SARS-CoV-2 Infection and COVID-19 Severity.

The SARS-CoV-2 infection was previously associated with the expression of the dopamine biosynthetic enzyme L-Dopa decarboxylase (DDC). Specifically, a negative correlation was detected between DDC mRNA and SARS-CoV-2 RNA levels in in vitro infected epithelial cells and the nasopharyngeal tissue of COVID-19 patients with mild/no symptoms. However, DDC, among other genes related to both DDC expression and SARS-CoV-2-infection (ACE2, dACE2, EPO), was upregulated in these patients, possibly attributed to an orchestrated host antiviral response. Herein, by comparing DDC expression in the nasopharyngeal swab samples of severe/critical to mild COVID-19 cases, we showed a 20 mean-fold reduction, highlighting the importance of the expression of this gene as a potential marker of COVID-19 severity. Moreover, we identified an association of SARS-CoV-2 infection with the expression of key catecholamine biosynthesis/metabolism-related genes, in whole blood samples from hospitalized patients and in cultured cells. Specifically, viral infection downregulated the biosynthetic part of the dopamine pathway (reduction in DDC expression up to 7.5 mean-fold), while enhanced the catabolizing part (increase in monoamine oxidases A and B expression up to 15 and 10 mean-fold, respectively) in vivo, irrespectively of the presence of comorbidities. In accordance, dopamine levels in the sera of severe cases were reduced (up to 3.8 mean-fold). Additionally, a moderate positive correlation between DDC and MAOA mRNA levels (r = 0.527, p < 00001) in the blood was identified upon SARS-CoV-2-infection. These observations were consistent to the gene expression data from SARS-CoV-2-infected Vero E6 and A549 epithelial cells. Furthermore, L-Dopa or dopamine treatment of infected cells attenuated the virus-derived cytopathic effect by 55% and 59%, respectively. The SARS-CoV-2 mediated suppression of dopamine biosynthesis in cell culture was, at least in part, attributed to hypoxia-like conditions triggered by viral infection. These findings suggest that L-Dopa/dopamine intake may have a preventive or therapeutic value for COVID-19 patients.

Multi-omics profiles refine L-dopa decarboxylase (DDC) as a reliable biomarker for prognosis and immune microenvironment of clear cell renal cell carcinoma.

Background: Increasing evidence indicates that L-dopa decarboxylase (DDC), which mediates aberrant amino acid metabolism, is significantly associated with tumor progression. However, the impacts of DDC are not elucidated clearly in clear cell renal cell carcinoma (ccRCC). This study aimed to evaluate DDC prognostic value and potential mechanisms for ccRCC patients. Methods: Transcriptomic and proteomic expressions of and clinical data including 532 patients with ccRCC (The Cancer Genome Atlas RNA-seq data), 226 ccRCC samples (Gene Expression Omnibus), 101 ccRCC patients from the E-MTAB-1980 cohort, and 232 patients with ccRCC with proteogenomic data (Fudan University Shanghai Cancer Center) were downloaded and analyzed to investigate the prognostic implications of DDC expression. Cox regression analyses were implemented to explore the effect of DDC expression on the prognosis of pan-cancer. The "limma" package identified the differentially expressed genes (DEGs) between high DDC subgroups and low DDC groups. Functional enrichments were performed based DEGs between DDC subgroups. The differences of immune cell infiltrations and immune checkpoint genes between DDC subgroups were analyzed to identify potential influence on immune microenvironment. Results: We found significantly decreased DDC expression in ccRCC tissues compared with normal tissues from multiple independent cohorts based on multi-omics data. We also found that DDC expression was correlated with tumor grades and stages.The following findings revealed that lower DDC expression levels significantly correlated with shorter overall survival (P <0.001) of patients with ccRCC. Moreover, we found that DDC expression significantly correlated with an immunosuppressive tumor microenvironment, higher intra-tumoral heterogeneity, elevated expression of immune checkpoint CD274, and possibly mediated malignant behaviors of ccRCC cells via the PI3k/Akt signaling pathway. Conclusion: The present study is the first to our knowledge to indicate that decreased DDC expression is significantly associated with poor survival and an immune-suppressive tumor microenvironment in ccRCC. These findings suggest that DDC could serve as a biomarker for guiding molecular diagnosis and facilitating the development of novel individual therapeutic strategies for patients with advanced ccRCC.

Dengue Virus Replication Is Associated with Catecholamine Biosynthesis and Metabolism in Hepatocytes.

Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression.

Association of Hepatitis C Virus Replication with the Catecholamine Biosynthetic Pathway.

A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.

Human L-Dopa decarboxylase interaction with annexin V and expression during apoptosis.

l-Dopa Decarboxylase (DDC) is a pyridoxal requiring enzyme that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (l-Dopa) to Dopamine (DA). The function of DDC in physiological and pathological biochemical pathways remains poorly understood, while the function and regulation of human DDC isoforms is almost completely elusive. We have shown that Annexin V, a fundamental apoptosis marker, is an inhibitor of l-Dopa decarboxylase activity. Here we show the interaction of both the full-length DDC and the truncated isoform alternative DDC (Alt-DDC) with Annexin V in human tissue and cell lines. Interestingly, DDC isoform expression is enhanced or remains unaffected following staurosporine (STS) treatment, despite increased levels of cytotoxicity and apoptosis. The findings presented here provide novel insights concerning the involvement of DDC in programmed cell death.

A Study for Therapeutic Treatment against Parkinson's Disease via Chou's 5-steps Rule.

The enzyme L-DOPA decarboxylase (DDC), also called aromatic-L-amino-acid decarboxylase, catalyzes the biosynthesis of dopamine, serotonin, and trace amines. Its deficiency or perturbations in expression result in severe motor dysfunction or a range of neurodegenerative and psychiatric disorders. A DDC substrate, L-DOPA, combined with an inhibitor of the enzyme is still the most effective treatment for symptoms of Parkinson's disease. In this review, we provide an update regarding the structures, functions, and inhibitors of DDC, particularly with regards to the treatment of Parkinson's disease. This information will provide insight into the pharmacological treatment of Parkinson's disease.

Emerging Role of l-Dopa Decarboxylase in Flaviviridae Virus Infections.

l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.

L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin.

L-Dopa decarboxylase (DDC) catalyzes the decarboxylation of L-Dopa to dopamine and 5-hydroxytryptophan (5-HTP) to serotonin. Although DDC has been purified from a variety of peripheral organs, including the liver, kidney and pancreas, the physiological significance of the peripherally expressed enzyme is not yet fully understood. DDC has been considered as a potential novel biomarker for various types of cancer, however, the role of DDC in the development of hepatocellular carcinoma (HCC) remains to be evaluated. Phosphatidylinositol 3-kinase (PI3K), on the other hand, has been shown to play a key role in the tumorigenesis, proliferation, metastasis, apoptosis, and angiogenesis of HCC by regulating gene expression. We initially identified the interaction of DDC with PI3K by means of the phage display methodology. This association was further confirmed in human hepatocellular carcinoma cell lines, human embryonic kidney cells, human neuroblastoma cells, as well as mouse brain, by the use of specific antibodies raised against DDC and PI3K. Functional aspects of the above interaction were studied upon treatment with the DDC inhibitor carbidopa and the PI3K inhibitor LY294002. Interestingly, our data demonstrate the expression of the neuronal type DDC mRNA in HCC cells. The present investigation provides new evidence on the possible link of DDC with the PI3K pathway, underlining the biological significance of this complex enzyme.

Dopamine synthesis and dopamine receptor expression are disturbed in recurrent miscarriages.

OBJECTIVES: l-dopa decarboxylase (DDC) is responsible for the synthesis of dopamine. Dopamine, which binds to the D2-dopamine receptor (D2R), plays an important role in the maintenance of pregnancy. Aim of our study was the analysis of DDC and D2R expression in placentas of spontaneous miscarriages (SMs) and recurrent miscarriages (RMs) in comparison to healthy controls. METHODS: Patients with SM (n = 15) and RM (n = 15) were compared with patients from healthy pregnancies (n = 15) (pregnancy weeks 7-13 each). Placental tissue has been collected from SMs and RMs from the first trimester (Department of Gynaecology and Obstetrics, LMU Munich) and from abruptions (private practice, Munich). Placental cell lines, BeWo- and JEG-3 cells, were stimulated with the trace amines T0AM and T1AM in vitro. RESULTS: Levels of DDC and D2R in trophoblasts and the decidua were lower in RMs in comparison to healthy controls. Stimulation of BeWo cells with T1AM significantly reduced DDC mRNA and protein levels. Via double-immunofluorescence, a DDC-positive cell type beneath decidual stromal cells and foetal EVT in the decidua could be detected. CONCLUSIONS: Downregulation of DDC and D2R in trophoblasts of RMs reflects a reduced signal cascade of catecholamines on the foetal side.

Increased trace amine-associated receptor 1 (TAAR1) expression is associated with a positive survival rate in patients with breast cancer.

PURPOSE: A correlation between breast cancer and thyroid disorders has been described in previous studies. Degraded thyroid hormones are referred to as trace amines. These endogenous amines have the ability to bind to the G-protein-coupled receptor TAAR1 (trace amine-associated receptor) and thereby activate it. TAAR1 is able to modulate the serotonergic and dopaminergic system in the brain and has so far been studied in the neurological field. The following study represents the first investigation of the regulation of TAAR1 in primary breast cancer (no metastases, M0). METHODS: Immunohistochemical analyses were carried out to detect TAAR1 expression in formalin fixed paraffin embedded breast cancer samples. Survival times of primary breast cancer patients (M0) with and without TAAR1 expression in their tumours were compared by Kaplan-Meier curves, and correlations between ordinal variables were determined with Spearman's rank correlation coefficient. RESULTS: The investigation showed a correlation between TAAR1 expression and tumour differentiation grade. A well differentiated tumour grade (G1) was associated with higher TAAR1 expression and HER2 and HER4 positivity predicted higher TAAR1 expression. A TAAR1 overexpression (IRS ≥ 6) was associated with significantly longer overall survival (OS) (p = 0.02) than that of reduced TAAR1 expression (IRS < 6) during a maximum follow-up of 14 years, demonstrating that TAAR1 has a favourable effect on OS of early breast cancer patients. CONCLUSIONS: We conclude that TAAR1 seems to be an independent predictor for breast cancer survival. Modulation of TAAR1 may represent a novel targeting strategy for breast cancer prevention and therapy.

L-DOPA decarboxylase mRNA levels provide high diagnostic accuracy and discrimination between clear cell and non-clear cell subtypes in renal cell carcinoma.

OBJECTIVES: Renal cell carcinoma (RCC) is the most frequent type of kidney cancer. RCC patients frequently present with arterial hypertension due to various causes, including intrarenal dopamine deficiency. L-DOPA decarboxylase (DDC) is the gene encoding the enzyme that catalyzes the biosynthesis of dopamine in humans. Several studies have shown that the expression levels of DDC are significantly deregulated in cancer. Thus, we herein sought to analyze the mRNA levels of DDC and evaluate their clinical significance in RCC. DESIGN AND METHODS: DDC levels were analyzed in 58 surgically resected RCC tumors and 44 adjacent non-cancerous renal tissue specimens via real-time PCR. Relative levels of DDC were estimated by applying the 2(-ΔΔC)T method, while their diagnostic accuracy and correlation with the clinicopathological features of RCC tumors were assessed by comprehensive statistical analysis. RESULTS: DDC mRNA levels were found to be dramatically downregulated (p<0.001) in RCC tumors, exhibiting remarkable diagnostic accuracy as assessed by ROC curve analysis (AUC: 0.910; p<0.001) and logistic regression (OR: 0.678; p=0.001). Likewise, DDC was found to be differentially expressed between clear cell RCC and the group of non-clear cell subtypes (p=0.001) consisted of papillary and chromophobe RCC specimens. Furthermore, a statistically significant inverse correlation was also observed when the mRNA levels of DDC were analyzed in relation to tumor grade (p=0.049). CONCLUSIONS: Our data showed that DDC constitutes a highly promising molecular marker for RCC, exhibiting remarkable diagnostic accuracy and potential to discriminate between clear cell and non-clear cell histological subtypes of RCC.

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target.

Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.