Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Role of Citrullus colocynthis and Psidium guajava Mediated Green Synthesized Silver Nanoparticles in Disease Resistance against Aeromonas hydrophila Challenge in Labeo rohita.

Green synthesis of metallic nanoparticles is an auspicious method of preparing nanoparticles using plant extracts that have lesser toxicity to animal cells and the host. In the present work, we analyzed the antibacterial activity of Citrullus colocynthis and Psidium guajava-mediated silver nanoparticles (Cc-AgNPs and Pg-AgNPs, respectively) against Aeromonas hydrophila (A. hydrophila) in an in vivo assay employing Labeo rohita (L. rohita). L. rohita were divided into six groups for both Cc-AgNPs and Pg-AgNPs treatments separately: Control, A. hydrophila infected, A. hydrophila + Ampicillin, A. hydrophila + Cc/Pg-AgNPs (25 µg/L), A. hydrophila + Cc/Pg-AgNPs (50 µg/L), and A. hydrophila + Cc/Pg-AgNPs (75 µg/L). Changes in different bio-indicators such as hematological, histological, oxidative stress, and cytokine analysis were observed. Interestingly, the infected fish treated with both types of AgNPs (Cc-AgNPs and Pg-AgNPs) exhibited a higher survival rate than the untreated infected fish and demonstrated signs of recovery from the infection, providing a compelling indication of the positive impact of phytosynthesized AgNPs. Disruptions in hematological and histological parameters were found in the infected fish. Both Cc-AgNPs and Pg-AgNPs showed recovery on the hematological and histological parameters. Analysis of oxidative stress and cytokine markers also revealed provoking evidence of the positive impact of Cc-AgNPs and Pg-AgNPs treatment against disease progression in the infected fish. The major finding of the study was that the higher concentrations of the nanoparticles (50 µg/L in the case of Cc-AgNPs and 75 µg/L in the case of Pg-AgNPs) were more effective in fighting against disease. In conclusion, our work presents novel insights for the use of green-synthesized AgNPs as economic and innocuous antibacterial candidates in aquaculture.

In-Vivo Bactericidal Potential of Mangifera indica Mediated Silver Nanoparticles against Aeromonas hydrophila in Cirrhinus mrigala.

The present study reports the green synthesis of silver nanoparticles from leaves' extract of Mangifera indica (M. indica) and their antibacterial efficacy against Aeromonas hydrophila (A. hydrophila) in Cirrhinus mrigala (C. mrigala). The prepared M. indica mediated silver nanoparticles (Mi-AgNPs) were found to be polycrystalline in nature, spherical in shapes with average size of 62 ± 13 nm. C. mrigala (n = ±15/group) were divided into six groups i.e., G1: control, G2: A. hydrophila challenged, G3: A. hydrophila challenged + Mi-AgNPs (0.01 mg/L), G4: A. hydrophila challenged + Mi-AgNPs (0.05 mg/L), G5: A. hydrophila challenged + Mi-AgNPs (0.1 mg/L) and G6: A. hydrophila challenged + M. indica extract (0.1 mg/L). Serum biochemical, hematological, histological and oxidative biomarkers were evaluated after 15 days of treatment. The liver enzyme activities, serum proteins, hematological parameters and oxidative stress markers were found to be altered in the challenged fish but showed retrieval effects with Mi-AgNPs treatment. The histological analysis of liver, gills and kidney of the challenged fish also showed regaining effects following Mi-AgNPs treatment. A CFU assay from muscle tissue provided quantitative data that Mi-AgNPs can hinder the bacterial proliferation in challenged fish. The findings of this work suggest that M. indica based silver nanoparticles can be promising candidates for the control and treatment of microbial infections in aquaculture.

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome.

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.

Citrullus colocynthis-Mediated Green Synthesis of Silver Nanoparticles and Their Antiproliferative Action against Breast Cancer Cells and Bactericidal Roles against Human Pathogens.

The present study investigated the biomedical potential of eco-friendly Citrullus colocynthis-mediated silver nanoparticles (Cc-AgNPs). The antibacterial efficacy of Cc-AgNPs was evaluated against two multidrug-resistant pathogenic bacterial strains, Escherichia coli and Pseudomonas aeruginosa. The antiproliferative and antilipidemic performance of the prepared particles was determined against the MCF7 cell line, a breast cancer cell line. The in vitro antibacterial assay revealed that Cc-AgNPs induced dose-dependent bactericidal activity, as a considerable increase in the zone of inhibition (ZOI) was noted at higher concentrations. Reduced proliferation, migration, spheroid size, and colony formation exhibited the substantial antiproliferative potential of Cc-AgNPs against MCF7 cells. Significant alterations in the expression of cell surface markers, apoptosis, and cell proliferation genes further confirmed the antiproliferative impact of Cc-AgNPs. Moreover, Cc-AgNPs exhibited antilipidemic activity by reducing cellular cholesterol and triglyceride levels and regulating key genes involved in lipogenesis. In conclusion, these results propose that Cc-AgNPs can be employed as a potent tool for future antibacterial and anticancer applications.

The role of the individual TOM subunits in the association of PINK1 with depolarized mitochondria.

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson's disease. PINK1, which is mutated in some cases of familial Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria. KEY MESSAGES: TOM70 is the main receptor for the import of PINK1 into mitochondria. TOM20 plays only a minor role in PINK1 recognition at the organellar outer membrane. PINK1 association with the TOM complex is reduced upon knock-down of TOM7. The lumen of the TOM pore is crucial for PINK1 association with the outer membrane. TcPINK1 blocks the TOM pore in depolarized mitochondria.

Selective localization of Mfn2 near PINK1 enables its preferential ubiquitination by Parkin on mitochondria.

Mutations in Parkin and PINK1 cause early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: (i) thioester intermediate formation on Parkin and (ii) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyses ubiquitin transfer to substrates. We found that His433 in the RING2 domain contributes to the catalysis of acyl transfer. In cells, the mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin (pUb) in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via pUb at these sites.

Mechanism of PINK1 activation by autophosphorylation and insights into assembly on the TOM complex.

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Risk of Covid-19 infection during endoscopy: Efficacy of personal protective equipment (PPE) in protecting health professionals.

OBJECTIVES: To correlate compliance to personal protective equipment (PPE) protocols and risk of exposure to SARS-COV-2 infection in endoscopy staff. METHODS: We included 85 endoscopic procedures performed at Lahore General Hospital from May to July 2020. Standard operating procedures (SOPs) were implemented for patient selection, risk stratification and personal protective equipment (PPE) use for endoscopy staff. Patient and endoscopy staff were followed for Covid-19 infection. PPE scores for staff and Covid-19 positivity on follow-up were correlated using student's t test. RESULTS: Following 85 procedures included, 2 (2.3%) patients became Covid-19 positive. PPE score was <9 in 5 (5.8%) procedures for endoscopist and Covid-19 developed in 2 (2.3%) of them, PPE score was <9 during 19 (22.3%) procedures in 1st assistant and 9 (10.5%) developed infection and for 2nd assistant PPE score was <9 in 19(22.3%) endoscopies and 5 (5.9%) tested positive for covid-19. Infectivity of endoscopy staff was 6.2%. Association between PPE score and risk of Covid-19 was not significant. (p value 0.51 for endoscopist, 0.10 for 1st assistant and 0.09 for 2nd assistant). CONCLUSION: Compliance of SOPs for infection control reduces risk of acquiring Covid-19 infection during endoscopy. Proper use of PPE is effective for safety of endoscopy staff.

Biosynthesis, characterization and anti-dengue vector activity of silver nanoparticles prepared from Azadirachta indica and Citrullus colocynthis.

We report here biosynthesis of silver nanoparticles (AgNPs) using aqueous extracts of (i) Azadirachta indica leaves and (ii) Citrullus colocynthis fruit and their larvicidal activity against Aedes aegypti. The UV-Vis spectroscopy absorption peaks occurred in the range of 412-416 nm for A. indica AgNPs and 416-431 nm for C. colocynthis AgNPs indicating the silver nature of prepared colloidal samples. The scanning electron microscopy examination revealed the spherical morphology of both types of NPs with average size of 17 ± 4 nm (A. indica AgNPs) and 26 ± 5 nm (C. colocynthis AgNPs). The X-ray diffraction pattern confirmed the face-centred cubic (FCC) structure with crystallite size of 11 ± 1 nm (A. indica AgNPs) and 15 ± 1 nm (C. colocynthis AgNPs) while characteristic peaks appearing in Fourier transform infrared spectroscopy analysis indicated the attachment of different biomolecules on AgNPs. The larvicidal activity at different concentrations of synthesized AgNPs (1-20 mg l-1) and extracts (0.5-1.5%) against Aedes aegypti was examined for 24 h. A concentration-dependent larvicidal potential of both types of AgNPs was observed. The LC50 values were found to be 0.3 and 1.25 mg l-1 for C. colocynthis AgNPs and A. indica AgNPs, respectively. However, both extracts did not exhibit any notable larvicidal activity.

Hepatocellular carcinoma occurs frequently and early after treatment in HCV genotype 3 infected persons treated with DAA regimens.

BACKGROUND: There are conflicting data regarding the risk of hepatocellular carcinoma (HCC) after direct-acting antiviral agent (DAA) treatment. Risk of HCC in HCV genotype-3 infected persons after DAA therapy is not well known. METHODS: We prospectively studied HCV infected persons initiated on a DAA regimen between October 2014 and March 2017 at two centers in Pakistan. All persons were free of HCC at study initiation. HCC was confirmed based on characteristic CT scan findings. Patients were followed for 12 months after the completion of therapy. RESULTS: A total of 662 persons initiated treatment. Median age (IQR) was 50 (41, 57) years and 48.8% were male. At baseline, 49.4% were cirrhotic, 91% were genotype 3 and 91.9% attained SVR. Treatment regimens used were: Sofosbuvir (SOF)/ribavirin (RBV)/pegylated interferon (PEG-IFN), 25.2%; SOF/RBV, 62.4%; SOF/RBV/daclatasavir (DCV), 10.6%; SOF/DCV, 2.0%. Incident HCC was detected in 42 patients (12.8%) in the 12-month period after treatment completion and was exclusively observed in those with cirrhosis. In multivariable Cox regression analysis, SVR was associated with a reduction in HCC risk (HR, 95% CI: 0.35, 0.14,0.85). In Kaplan-Meier plots by treatment regimen, those treated with SOF/RBV, SOF/RBV/DCV, or SOF/DCV regimens had a shorter HCC-free survival compared with those treated with a SOF/RBV/PEG-IFN regimen. CONCLUSION: In a predominantly genotype 3 cohort, incident HCC occurred frequently and early after treatment completion, and exclusively in those with pre-treatment cirrhosis. SVR reduced the risk of HCC. Treating HCV infected persons before development of cirrhosis may reduce risk of HCC.

Significance of Testing Anti-HBcIgM Antibodies for the Screening of Hepatitis B in the Donor Blood.

BACKGROUND: The purpose to perform this study was to screen blood donors for possible occult HBV by checking the seroprevalence of the hepatitis B antibodies in blood donors. It was a Cross-sectional study conducted at Blood Bank of Lahore General Hospital Lahore from April to June 2015 (3-months). METHODS: In this prospective study, 180 healthy blood donors, presenting to the blood bank of Lahore General Hospital were selected. Their detailed demographic data and blood samples were collected. HBsAg testing was done by ELISA and further HBc IgM testing was also done by ELISA. Those testing positive for HBc IgM were further evaluated by real-time PCR to detect HBV DNA. RESULTS: Mean duration of the life span was 26.51 years with a range of 18-61 years. Sex distribution show 93.9% (n=169) males and 6.1% (n=11) females. HBsAg was positive in 3.3% (n=6) while their HBc IgM was negative and HBc IGM was positive in 2.2% (n=4) of the healthy donors in whom HBsAg was found negative by ICT method. further qualitative HBV DNA by rt-PCR was done on those positive with anti HBc IgM and no patient had HBV DNA detected from their blood. CONCLUSION: Without routine screening of the sera for the HBc Antibody, the low-level HBV viraemia may not be detected as the nonappearance of the surface antigen in the blood of apparently healthy donors do not ensure the absence of circulating virus in the blood of these donors.

New insights into the structure of PINK1 and the mechanism of ubiquitin phosphorylation.

Mutations in PINK1 cause early-onset recessive Parkinson's disease. This gene encodes a protein kinase implicated in mitochondrial quality control via ubiquitin phosphorylation and activation of the E3 ubiquitin ligase Parkin. Here, we review and analyze functional features emerging from recent crystallographic, nuclear magnetic resonance (NMR) and mass spectrometry studies of PINK1. We compare the apo and ubiquitin-bound PINK1 structures and reveal an allosteric switch, regulated by autophosphorylation, which modulates substrate recognition. We critically assess the conformational changes taking place in ubiquitin and the Parkin ubiquitin-like domain in relation to its binding to PINK1. Finally, we discuss the implications of these biophysical findings in our understanding of the role of PINK1 in mitochondrial function, and analyze the potential for structure-based drug design.

PINK1 autophosphorylation is required for ubiquitin recognition.

Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.

Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.

A Ubl/ubiquitin switch in the activation of Parkin.

Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.

Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population.

BACKGROUND: Oculocutaneous albinism (OCA) is caused by a group of genetically heterogeneous inherited defects that result in the loss of pigmentation in the eyes, skin and hair. Mutations in the TYR, OCA2, TYRP1 and SLC45A2 genes have been shown to cause isolated OCA. No comprehensive analysis has been conducted to study the spectrum of OCA alleles prevailing in Pakistani albino populations. METHODS: We enrolled 40 large Pakistani families and screened them for OCA genes and a candidate gene, SLC24A5. Protein function effects were evaluated using in silico prediction algorithms and ex vivo studies in human melanocytes. The effects of splice-site mutations were determined using an exon-trapping assay. RESULTS: Screening of the TYR gene revealed four known (p.Arg299His, p.Pro406Leu, p.Gly419Arg, p.Arg278*) and three novel mutations (p.Pro21Leu, p.Cys35Arg, p.Tyr411His) in ten families. Ex vivo studies revealed the retention of an EGFP-tagged mutant (p.Pro21Leu, p.Cys35Arg or p.Tyr411His) tyrosinase in the endoplasmic reticulum (ER) at 37°C, but a significant fraction of p.Cys35Arg and p.Tyr411His left the ER in cells grown at a permissive temperature (31°C). Three novel (p.Asp486Tyr, p.Leu527Arg, c.1045-15 T > G) and two known mutations (p.Pro743Leu, p.Ala787Thr) of OCA2 were found in fourteen families. Exon-trapping assays with a construct containing a novel c.1045-15 T > G mutation revealed an error in splicing. No mutation in TYRP1, SLC45A2, and SLC24A5 was found in the remaining 16 families. Clinical evaluation of the families segregating either TYR or OCA2 mutations showed nystagmus, photophobia, and loss of pigmentation in the skin or hair follicles. Most of the affected individuals had grayish-blue colored eyes. CONCLUSIONS: Our results show that ten and fourteen families harbored mutations in the TYR and OCA2 genes, respectively. Our findings, along with the results of previous studies, indicate that the p.Cys35Arg, p.Arg278* and p.Gly419Arg alleles of TYR and the p.Asp486Tyr and c.1045-15 T > G alleles of OCA2 are the most common causes of OCA in Pakistani families. To the best of our knowledge, this study represents the first documentation of OCA2 alleles in the Pakistani population. A significant proportion of our cohort did not have mutations in known OCA genes. Overall, our study contributes to the development of genetic testing protocols and genetic counseling for OCA in Pakistani families.