Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia.

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here, we present evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. Our findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

FBXO22 inhibits proliferation and metastasis of cervical cancer cells by mediating ubiquitination-dependent degradation of GAK.

Cervical cancer is one of the recognized malignant tumors of female reproductive system. At present, the research and development of biomarkers has attracted increasing attention, and the wide application of clinical cervical cancer screening strategies has significantly reduced its morbidity and mortality. A member of the F-box protein family, FBXO22, is involved in cell cycle, DNA damage repair and many other processes. Dysregulation of FBXO22 plays an important role in the occurrence and development of various tumors, including ovarian cancer, liver cancer and lung cancer. Nevertheless, the effect of FBXO22 in cervical cancer needs further investigation. We found that FBXO22 inhibited cervical cancer cell proliferation, migration and invasion. The results of proteomics studies suggested FBXO22 appears to target the Cyclin G Associated Kinase (GAK) for degradation. The combined results of analysis of cultured cells with altered abundance of FBXO22 by depletion or over-expression in the presence or absence of proteasomal inhibitor, comparison of protein decay rate, as well as cellular ubiquitination, support a hypothesis that FBXO22 mediates the ubiquitin-dependent degradation of GAK. Taken together, our data suggest that FBXO22 has a protective role in cervical cancer.

Synthesis of a 3,7-Disubstituted Isothiazolo[4,3-b]pyridine as a Potential Inhibitor of Cyclin G-Associated Kinase.

Disubstituted isothiazolo[4,3-b]pyridines are known inhibitors of cyclin G-associated kinase. Since 3-substituted-7-aryl-isothiazolo[4,3-b]pyridines remain elusive, a strategy was established to prepare this chemotype, starting from 2,4-dichloro-3-nitropyridine. Selective C-4 arylation using ligand-free Suzuki-Miyaura coupling and palladium-catalyzed aminocarbonylation functioned as key steps in the synthesis. The 3-N-morpholinyl-7-(3,4-dimethoxyphenyl)-isothiazolo[4,3-b]pyridine was completely devoid of GAK affinity, in contrast to its 3,5- and 3,6-disubstituted congeners. Molecular modeling was applied to rationalize its inactivity as a GAK ligand.

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Utilizing comprehensive and mini-kinome panels to optimize the selectivity of quinoline inhibitors for cyclin G associated kinase (GAK).

We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.

Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK).

SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 µM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Impact of Parkinson's disease risk loci on age at onset.

BACKGROUND: The aim of this study was to assess whether recently identified Parkinson's disease (PD) risk genes also influence age at onset in PD. METHODS: We genotyped 23 single-nucleotide polymorphisms in 1,526 Danish PD patients and performed linear regression analyses with age at onset. The combined impact of PD risk loci on age at onset was assessed by linear regression analyses using a weighted genetic risk score. RESULTS: The strongest effects were observed with rs12726330 in GBA (beta = -3.63, P = 2.0 × 10(-5) ) and rs34311866 in TMEM175/GAK (beta = -1.19, P = 4.0 × 10(-3) ), corresponding to a 3.6-y and 1.2-y decrease of age at onset per risk allele, respectively. The weighted genetic risk score yielded significant association with reduced onset age (P = 3.98 × 10(-3) ), although the variance explained was small (0.6%), and the effect was mostly driven by polymorphisms in GBA and TMEM175/GAK. CONCLUSIONS: Overall, our study indicates that GBA and TMEM175/GAK significantly alter age at onset in PD.

A new regulator of autophagy initiation in glia.

Macroautophagy/autophagy is the major degradation pathway in neurons for eliminating damaged proteins and organelles in Parkinson disease (PD). Like neurons, glial cells are important contributors to PD, yet how autophagy is executed in glia and whether it is using similar interplay as in neurons or other tissues, remain largely elusive. Recently, we reported that the PD risk factor, GAK/aux (cyclin-G-associated kinase/auxilin), regulates the onset of glial autophagy. In the absence of GAK/aux, the number and size of the autophagosomes and autophagosomal precursors increase in adult fly glia and mouse microglia. The protein levels of components in the initiation and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes are generally upregulated. GAK/aux interacts with the master initiation regulator ULK1/Atg1 (unc-51 like autophagy activating kinase 1) via its uncoating domain, hinders autophagy activation by competing with ATG13 (autophagy related 13) for binding to the ULK1 C terminus, and regulates ULK1 trafficking to phagophores. Nonetheless, lack of GAK/aux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/aux might play additional roles. Overall, our findings reveal a new regulator of autophagy initiation in glia, advancing our understanding on how glia contribute to PD in terms of eliminating pathological protein aggregates.Abbreviations: ATG13: autophagy related 13; GAK/aux: cyclin G associated kinase/auxilin; PtdIns3K: phosphatidylinositol 3-kinase; PD: Parkinson disease; ULK1/Atg1: unc-51 like autophagy activating kinase 1.

Naturally selected rilpivirine-resistant HIV-1 variants by host cellular immunity.

BACKGROUND: Rilpivirine is listed as an alternative key drug in current antiretroviral therapy (ART) guidelines. E138G/A/K in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rilpivirine resistance-associated mutations and can be identified in a few ART-naive patients, although at low frequency. The 138th position in HIV-1 RT is located in one of the putative epitopes of human leukocyte antigen (HLA)-B*18-restricted cytotoxic T lymphocytes (CTLs). CTL-mediated immune pressure selects escape mutations within the CTL epitope. Here we tested whether E138G/A/K could be selected by HLA-B*18-restricted CTLs. METHODS: The amino acid variation at the 138th position was compared between ART-naive HIV-1-infected patients with and without HLA-B*18. The optimal epitope containing the 138th position was determined and the impact of E138G/A/K on CTL response was analyzed by epitope-specific CTLs. The effect of E138G/A/K on drug susceptibility was determined by constructing recombinant HIV-1 variants. RESULTS: The prevalence of E138G/A/K was 21% and 0.37% in 19 and 1088 patients with and without HLA-B*18, respectively (odds ratio, 72.3; P = 4.9 × 10(-25)). The CTL response was completely abolished by the substitution of E138G/A/K in the epitope peptide. E138G/A/K conferred 5.1-, 7.1-, and 2.7-fold resistance to rilpivirine, respectively. CONCLUSIONS: E138G/A/K can be selected by HLA-B*18-restricted CTLs and confer significant rilpivirine resistance. We recommend drug resistance testing before the introduction of rilpivirine-based ART in HLA-B*18-positive patients.

Reaffirmation of GAK, but not HLA-DRA, as a Parkinson's disease susceptibility gene in a Taiwanese population.

Recent genome-wide association studies of Parkinson's disease (PD) in Caucasian populations have identified two new susceptibility loci, GAK and HLA-DRA; however, only limited information exists regarding the involvement of these genes in PD risk in other ethnic groups. Here, we examined whether these genetic effects were consistent in a Taiwanese PD population. In a total 900 participants, including 448 PD patients and 452 control subjects, we genotyped the rs11248051 and rs1564282 variants of GAK, and the rs3129882 variant of HLA-DRA. Logistic regression analysis was used to test for associations between genotype and PD under an additive model, adjusting for age and gender. Subjects with CT/TT genotypes of GAK rs11248051 had a modestly increased association with PD compared to those with CC genotype (OR = 1.37; 95% CI = 1.09, 1.87; P = 0.03). Carriers and non-carriers exhibited indistinguishable phenotypes in regards to clinical presentation and onset age. We observed no association between PD risk and GAK rs1564282 or HLA-DRA rs3129882 variant. The different genetic effects between Taiwanese and Caucasian populations may come from differences in population structure and geographic region-specific genetic-environmental interactions. In conclusion, our results supported the association between the rs11248051 variant in GAK and PD risk in a Taiwanese population. Future functional studies of GAK in neuronal degeneration are warranted to unravel its role in the pathogenetic mechanism of PD.

Design, synthesis, and biological evaluation of novel ruxolitinib and baricitinib analogues for potential use against COVID-19.

The coronavirus pandemic known as COVID-19 caused by severe acute respiratory syndrome coronavirus 2, threatens public health worldwide. Approval of COVID-19 vaccines and antiviral drugs have greatly reduced the severe cases and mortality rate. However, the continuous mutations of viruses are challenging the efficacies of vaccines and antiviral drugs. A drug repurposing campaign has identified two JAK1/2 inhibitors ruxolitinib and baricitinib as potential antiviral drugs. Ruxolitinib and baricitinib exert dual antiviral effect by modulation of inflammatory response via JAK1/2 and inhibition of viral entry via AAK1 and GAK. Inspired by this, in an effort to diversify chemical space, three analogues ((R)-8, (S)-8, and 9) of ruxolitinib and baricitinb were made using a scaffold hopping strategy. Compound 9 displayed potent and comparable potencies against AAK1, JAK1, and JAK2 compared to baricitinib. Notably, compound 9 showed better selectivity for AAK1, JAK1, and JAK2 over GAK. Besides, compound 9 displayed good druglikeness according to Lipinski's and Veber's rule. We thereby identified a potential lead compound 9, which might be used for the further development of anti-coronaviral therapy.

The cyclin G-associated kinase (GAK) inhibitor SGC-GAK-1 inhibits neurite outgrowth and synapse formation.

Protein kinases are responsible for protein phosphorylation and are involved in important signal transduction pathways; however, a considerable number of poorly characterized kinases may be involved in neuronal development. Here, we considered cyclin G-associated kinase (GAK) as a candidate regulator of neurite outgrowth and synaptogenesis by examining the effects of the selective GAK inhibitor SGC-GAK-1. SGC-GAK-1 treatment of cultured neurons reduced neurite length and decreased synapse number and phosphorylation of neurofilament 200-kDa subunits relative to the control. In addition, the related kinase inhibitor erlotinib, which has distinct specificity and potency from SGC-GAK-1, had no effect on neurite growth, unlike SGC-GAK-1. These results suggest that GAK may be physiologically involved in normal neuronal development, and that decreased GAK function and the resultant impaired neurite outgrowth and synaptogenesis may be related to neurodevelopmental disorders.

New regulators of PRKN-independent mitophagy.

Mitophagy, a type of selective autophagy targeting damaged or superfluous mitochondria, is critical to maintain cell homeostasis. Besides the well-characterized PRKN-dependent mitophagy, PRKN-independent mitophagy also plays significant physiological roles. In a recent study, researchers from Anne Simonsen's lab discovered two lipid binding kinases, GAK and PRKCD, as positive regulators of PRKN-independent mitophagy. The researchers further investigated how these two proteins regulate mitophagy and demonstrated their roles in vivo. Focusing on the less known PRKN-independent mitophagy regulators, these findings shed light on understanding the mechanism of mitophagy and its relation to diseases.

GAK and PRKCD kinases regulate basal mitophagy.

The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal "housekeeping" mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings.Abbreviations: CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; DFP: deferiprone; GAK: cyclin G associated kinase; HIF1A: hypoxia inducible factor 1 subunit alpha; PRKC/PKC: protein kinase C; PRKCD: protein kinase C delta; PRKN: parkin RBR E3 ubiquitin protein ligase.

Association of GAK rs1564282 With Susceptibility to Parkinson's Disease in Chinese Populations.

The susceptibility of the GAK rs1564282 variant in Parkinson's disease (PD) in Europeans was identified using a series of published genome-wide association studies. Recently, some studies focused on the association between rs1564282 and PD risk in Chinese populations but with inconsistent results. Thus, we conducted an updated meta-analysis with a total of 7,881 samples (4,055 PD cases and 3,826 controls) from eligible studies. After excluding significant heterogeneity, we showed that the rs1564282 variant was significantly associated with PD in Chinese populations (p = 1.00E-04, odds ratio = 1.28 and 95% confidence interval = 1.16-1.42). The sensitivity analysis showed that the association between rs1564282 and PD was not greatly influenced, and there was no significant publication bias among the included studies. Consequently, this meta-analysis indicates that the GAK rs1564282 variant is significantly associated with susceptibility to PD in Chinese populations.

Roles of existing drug and drug targets for COVID-19 management.

In December 2019, a highly transmissible, pneumonia epidemic caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), erupted in China and other countries, resulting in devastation and health crisis worldwide currently. The search and using existing drugs support to curb the current highly contagious viral infection is spirally increasing since the pandemic began. This is based on these drugs had against other related RNA-viruses such as MERS-Cov, and SARS-Cov. Moreover, researchers are scrambling to identify novel drug targets and discover novel therapeutic options to vanquish the current pandemic. Since there is no definitive treatment to control Covid-19 vaccines are remain to be a lifeline. Currently, many vaccine candidates are being developed with most of them are reported to have positive results. Therapeutic targets such as helicases, transmembrane serine protease 2, cathepsin L, cyclin G-associated kinase, adaptor-associated kinase 1, two-pore channel, viral virulence factors, 3-chymotrypsin-like protease, suppression of excessive inflammatory response, inhibition of viral membrane, nucleocapsid, envelope, and accessory proteins, and inhibition of endocytosis were identified as a potential target against COVID-19 infection. This review also summarizes plant-based medicines for the treatment of COVID-19 such as saposhnikoviae divaricata, lonicerae japonicae flos, scutellaria baicalensis, lonicera japonicae, and some others. Thus, this review aimed to focus on the most promising therapeutic targets being repurposed against COVID-19 and viral elements that are used in COVID-19 vaccine candidates.

Effect of Silicate and Phosphate Solubilizing Rhizobacterium Enterobacter ludwigii GAK2 on Oryza sativa L. under Cadmium Stress.

Silicon and phosphorus are elements that are beneficial for plant growth. Despite the abundant availability of silicate and phosphate in the Earth's crust, crop nutritional requirements for silicon and phosphorus are normally met through the application of fertilizer. However, fertilizers are one of the major causes of heavy metal pollution. In our study, we aimed to assess silicate and phosphate solubilization by the bacteria Enterobacter ludwigii GAK2, in the presence and absence of phosphate [Ca3(PO4)2] or silicate (Mg2O8Si3), to counteract cadmium stress in rice (Oryza sativa L). Our results showed that the GAK2-treated rice plants, grown in soil amended with phosphate [Ca3(PO4)2] or silicate (Mg2O8Si3), had significantly reduced cadmium content, and enhanced plant growth promoting characteristics including fresh shoot and root weight, plant height, and chlorophyll content. These plants showed significant downregulation of the cadmium transporter gene, OsHMA2, and upregulation of the silicon carrier gene, OsLsi1. Moreover, jasmonic acid levels were significantly reduced in the GAK2-inoculated plants, and this was further supported by the downregulation of the jasmonic acid related gene, OsJAZ1. These results indicate that Enterobacter ludwigii GAK2 can be used as a silicon and phosphorus bio-fertilizer, which solubilizes insoluble silicate and phosphate, and mitigates heavy metal toxicity in crops.

Targeting the Water Network in Cyclin G-Associated Kinase (GAK) with 4-Anilino-quin(az)oline Inhibitors.

Water networks within kinase inhibitor design and more widely within drug discovery are generally poorly understood. The successful targeting of these networks prospectively has great promise for all facets of inhibitor design, including potency and selectivity for the target. Herein, we describe the design and testing of a targeted library of 4-anilinoquin(az)olines for use as inhibitors of cyclin G-associated kinase (GAK). GAK cellular target engagement assays, ATP binding-site modelling and extensive water mapping provide a clear route to access potent inhibitors for GAK and beyond.