Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b‐ and AP4‐dependent pathway

The intracellular trafficking and proteolytic processing of the membrane‐bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid‐beta (Aβ) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by β‐secretase (BACE1) and γ‐secretase. Here, we show that the majority of newly synthesized APP is transported from the trans‐Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post‐Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aβ secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aβ.      

RAD51AP1 promotes progression of ovarian cancer via TGF-β/Smad signalling pathway

Ovarian cancer (OC) is one of the leading causes of female deaths. However, the molecular pathogenesis of OC has still remained elusive. This study aimed to explore the potential genes associated with the progression of OC. In the current study, 3 data sets of OC were downloaded from the GEO database to identify hub gene. Somatic mutation data obtained from TCGA were used to analyse the mutation. Immune cells were used to estimate effect of the hub gene to the tumour microenvironment. RNA-seq and clinical data of OC patients retrieved from TCGA were used to investigate the diagnostic and prognostic values of hub gene. A series of in vitro assays were performed to indicate the function of hub gene and its possible mechanisms in OC. As a result, RAD51AP1 was found as a hub gene, which expression higher was mainly associated with poor survival in OC patients. Up-regulation of RAD51AP1 was closely associated with mutations. RAD51AP1 up-regulation accompanied by accumulated Th2 cells, but reduced CD4 + T cells and CD8 + T cells. Nomogram demonstrated RAD51AP1 increased the accuracy of the model. Down-regulation of RAD51AP1 suppressed proliferation, migration and invasion capabilities of OC cells in vitro. Additionally, scatter plots showed that RAD51AP1 was positively correlated with genes in TGF-β/Smad pathway. The above-mentioned results were validated by RT-qPCR and Western blotting. In conclusion, up-regulation of RAD51AP1 was closely associated with mutations in OC. RAD51AP1 might represent an indicator for predicting OS of OC patients. Besides, RAD51AP1 might accelerate progression of OC by TGF-β/Smad signalling pathway.     

Molecular level damages of low power pulsed laser radiation in a marine bacterium Pseudoalteromonas carrageenovora

AIM: To study the molecular level damages in a marine bacterium, Pseudoalteromonas carrageenovora, exposed to low power pulsed laser radiation from an Nd:YAG laser. METHODS AND RESULTS: The laser damages in bacterial DNA were monitored by studying the formation of apurinic/apyrimidinic (AP) sites. Molecular probe kits were used for this purpose. Occurrence of lesions in the cell walls was monitored under a transmission electron microscope (TEM). The results showed that laser radiation significantly increased the number of AP sites in the bacterial DNA. This increase corresponded to the laser fluence (J cm(-2)) and to the duration of laser irradiation. TEM observation showed the occurrence of lesions in bacterial cell walls upon laser irradiation. CONCLUSIONS: It is concluded that bacteria exposed to laser irradiation suffers DNA damages and resulted in broken cell walls. These events led to bacterial mortality. These are in addition to the mechanisms reported earlier such as the photochemical reactions occurring inside the cells upon exposure to low power laser. SIGNIFICANCE AND IMPACT OF THE STUDY: These results help us to understand the mechanisms of bacterial mortality on exposure to low power pulsed laser irradiation and are useful in formulating a laser treatment strategy to kill bacteria.     

Human melanoma TrkC: its association with a purine-analog-sensitive kinase activity

The various members of the Trk tyrosine kinase family and p75 neurotrophin receptor (p75(NTR)) have been identified as signaling receptors for the structurally related members of the neurotrophins (NT) family. We have previously reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of extracellular-matrix degradative enzymes. These cells express aberrant levels of functional p75(NTR) and TrkC, the putative high-affinity receptor for the neurotrophin NT-3. Here we demonstrate that, by using sensitive immune-complex kinase assays in human brain-metastatic (70W) melanoma cells, TrkC receptors associate with a kinase activity exhibiting a dose-dependent susceptibility to inhibition by the purine-analogs 6-thioguanine and 2-aminopurine. The activity of this purine-analog-sensitive kinase (PASK) was induced by NT-3 in a time-dependent fashion, phosphorylating exogenous myelin basic protein (MBP) but not denatured enolase. It is similar to the one reported to relate with p75(NTR) and TrkA receptors and stimulated by the prototypic NT, nerve growth factor. Thus, PASKs may represent unique signaling components common to NT receptors that could engage joint downstream signaling effectors in brain-metastatic melanoma.     

A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bak-independent apoptosis

We have previously shown that the natural diterpenoid derivative S3 induced Bim upregulation and apoptosis in a Bax/Bak-independent manner. However, the exact molecular target(s) of S3 and the mechanism controlling Bim upregulation are still not clear. Here, we identify that S3 targets the selenoproteins TrxR1 and TrxR2 at the selenocysteine residue of the reactive center of the enzymes and inhibits their antioxidant activities. Consequently, cellular ROS is elevated, leading to the activation of FOXO3a, which contributes to Bim upregulation in Bax/Bak-deficient cells. Moreover, S3 retards tumor growth in subcutaneous xenograft tumors by inhibiting TrxR activity in vivo. Our studies delineate the signaling pathway controlling Bim upregulation, which results in Bax/Bak-independent apoptosis and provide evidence that the compounds can act as anticancer agents based on mammalian TrxRs inhibition.     

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA.     

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3′ → 5′ exonuclease activities. However, it remains unclear whether these enzymes hold 3′-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3′-repair phosphodiesterase and 3′-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5 mM MnCl₂ at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37°C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3′-blocking sugar-phosphate and 3′-phosphate moieties at DNA strand breaks with an extremely high efficiency (k_cat/K_M = 440 and 1280  μM^-1∙min⁻¹, respectively), while MtbNfo exhibits much lower 3′-repair activities (k_cat/K_M = 0.26 and 0.65 μM^-1∙min⁻¹, respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H₂O₂ to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host- imposed factors.