Cloud observation serves as the fundamental bedrock for acquiring comprehensive cloud-related information. The categorization of distinct ground-based clouds holds profound implications within the ...meteorological domain, boasting significant applications. Deep learning has substantially improved ground-based cloud classification, with automated feature extraction being simpler and far more accurate than using traditional methods. A reengineering of the DenseNet architecture has given rise to an innovative cloud classification method denoted as CloudDenseNet. A novel CloudDense Block has been meticulously crafted to amplify channel attention and elevate the salient features pertinent to cloud classification endeavors. The lightweight CloudDenseNet structure is designed meticulously according to the distinctive characteristics of ground-based clouds and the intricacies of large-scale diverse datasets, which amplifies the generalization ability and elevates the recognition accuracy of the network. The optimal parameter is obtained by combining transfer learning with designed numerous experiments, which significantly enhances the network training efficiency and expedites the process. The methodology achieves an impressive 93.43% accuracy on the large-scale diverse dataset, surpassing numerous published methods. This attests to the substantial potential of the CloudDenseNet architecture for integration into ground-based cloud classification tasks.
Advancement in our knowledge of deubiquitinases (DUBs) and their biological functions requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions. ...Activity-based DUB probes (DUB ABPs) have been widely used in investigating the function and activity of DUBs. However, most ubiquitin (Ub)-based DUB ABPs are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs and DUB complexes. Here, we report a new class of cell-permeable DUB ABPs that enable intracellular DUB profiling. We used a semisynthetic approach to generate modular ubiquitin-based DUB probes containing a reactive warhead for covalent trapping of DUBs with a catalytic cysteine. We employed cell-penetrating peptides (CPPs), particualrly cyclic polyarginine (cR10), to deliver the DUB ABPs into cells, as confirmed using live-cell fluorescence microscopy and DUB ABPs containing a fluorophore at the C-terminus of Ub. In comparison to TAT, enhanced intacellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cell-permeable DUB ABPs, we carried out DUB profiling in intact HeLa cells, and identified active DUBs using immunocapture and label-free quantitative mass spectrometry. Additionally, we demonstrated that the cell-permeable DUB ABPs can be used in assessing the inhibition of DUBs by small-molecule inhibitors in intact cells. Our results indicate that cell-permeable DUB ABPs hold great promise in providing a better understanding of the cellular functions of DUBs and advancing drug discovery efforts targeting human DUBs.
The rapid growth in ubiquitin biology requires facile chemical approaches for protein ubiquitylation that can overcome the common problem of low yield faced by the enzymatic reaction catalyzed by ...ubiquitin ligases. We report a chemical approach for monoubiquitylation and SUMOylation of PCNA through disulfide exchange and intein chemistry. We used the chemically ubiquitylated and SUMOylated PCNAs in studying translesion DNA synthesis and revealed a surprising degree of flexibility of the ubiquitin modification.
Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have ...been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
To solve the problem of feature distribution discrepancy in cross-corpus speech emotion recognition tasks, this paper proposed an emotion recognition model based on multi-task learning and subdomain ...adaptation, which alleviates the impact on emotion recognition. Existing methods have shortcomings in speech feature representation and cross-corpus feature distribution alignment. The proposed model uses a deep denoising auto-encoder as a shared feature extraction network for multi-task learning, and the fully connected layer and softmax layer are added before each recognition task as task-specific layers. Subsequently, the subdomain adaptation algorithm of emotion and gender features is added to the shared network to obtain the shared emotion features and gender features of the source domain and target domain, respectively. Multi-task learning effectively enhances the representation ability of features, a subdomain adaptive algorithm promotes the migrating ability of features and effectively alleviates the impact of feature distribution differences in emotional features. The average results of six cross-corpus speech emotion recognition experiments show that, compared with other models, the weighted average recall rate is increased by 1.89~10.07%, the experimental results verify the validity of the proposed model.
Activity-based diubiquitin probes are highly useful in probing the deubiquitinase (DUB) activity and ubiquitin chain linkage specificity. Here we describe a detailed protocol to synthesize a new ...class of diubiquitin DUB probes. In this method, two ubiquitin moieties are connected through a linker resembling the native linkage in size and containing a Michael acceptor for trapping the DUB active-site cysteine. Detailed procedures for generating the linker molecule are also described.
Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and ...polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.
Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellular processes. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating ...enzymes (DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinated proteins as substrates, more direct evidence is needed to support this notion. Here we report protein-targeting activity-based DUB probes that allowed the identification of DUBs recognizing monoubiquitinated proliferating cell nuclear antigen (PCNA) in
. This new class of DUB probes contain a Michael acceptor as a warhead between ubiquitin and the target protein PCNA through a linkage that mimics the native isopeptide bond. We selected two known and biologically relevant ubiquitination sites on PCNA to generate the DUB probes. This allowed us to interrogate the site-specific deubiquitination of a target protein by DUBs. DUBs were profiled in yeast cell lysates using the two Ub-PCNA DUB probes in conjunction with two control probes that contain a noncleavable linkage but no warhead. We identified yeast DUBs through pulldown coupled with quantitative mass spectrometry analysis of the pulled down proteins. Our results showed that specific yeast DUBs recognize monoubiquitinated PCNA and corroborated previous genetic study. We also identified DUBs as potential new deubiquitinase of PCNA. Remarkably, identified DUBs clearly distinguish the different modification sites on PCNA, thus supporting a high level of DUB specificity beyond the target protein identity.
Ubiquitination is an important protein post-translational modification regulating many cellular processes in eukaryotes. Ubiquitination is catalyzed by a three-enzyme cascade resulting in the ...conjugation of the C-terminal carboxylate of ubiquitin (Ub) to the -amino group of a lysine residue in the acceptor protein
via
an isopeptide bond.
In vitro
enzymatic ubiquitination utilizing Ub ligases has been successfully employed to generate Ub dimers and polymers. However, limitations of the enzymatic approach exist, particularly due to the requirement of specific Ub ligase for any given target protein and the low catalytic efficiency of the Ub ligase. To achieve an in-depth understanding of the molecular mechanism of Ub signaling, new methods are needed to generate mono- and poly-ubiquitinated proteins at a specific site with defined polyubiquitin chain linkage and length. Chemical methods offer an attractive solution to the above-described challenges. In this review, we summarize the recently developed chemical methods for generating ubiquitinated proteins using synthetic and semisynthetic approaches. These new tools and approaches, as an important part of the Ub toolbox, are crucial to our understanding and exploitation of the Ub system for novel therapeutics.
Chemical methods for protein site-specific ubiquitination are important for the understanding of Ub signaling.
To ensure efficient and timely replication of genomic DNA, organisms in all three kingdoms of life possess specialized translesion DNA synthesis (TLS) polymerases (Pols) that tolerate various types ...of DNA lesions. It has been proposed that an exchange between the replicative DNA Pol and the TLS Pol at the site of DNA damage enables lesion bypass to occur. However, to date the molecular mechanism underlying this process is not fully understood. In this study, we demonstrated in a reconstituted system that the exchange of Saccharomyces cerevisiae Polδ with Polη requires both the stalling of the holoenzyme and the monoubiquitination of proliferating cell nuclear antigen (PCNA). A moving Polδ holoenzyme is refractory to the incoming Polη. Furthermore, we showed that the Polη C-terminal PCNA-interacting protein motif is required for the exchange process. We also demonstrated that the second exchange step to bring back Polδ is prohibited when Lys-164 of PCNA is monoubiquitinated. Thus the removal of the ubiquitin moiety from PCNA is likely required for the reverse exchange step after the lesion bypass synthesis by Polη.
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