Mediated Protein Degradation Research Articles

The TP53-activated E3 ligase RNF144B is a tumour suppressor that prevents genomic instability.

TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes have been extensively studied, others, like the recently identified RNF144B, remained poorly understood. This E3 ubiquitin ligase has shown potent tumor suppressor activity in murine Eμ Myc-driven lymphoma, emphasizing its significance in the TP53 network. However, little is known about its targets and its role in cancer development, requiring further exploration. In this work, we investigate RNF144B's impact on tumor suppression beyond the hematopoietic compartment in human cancers. Employing TP53 wild-type cells, we generated models lacking RNF144B in both non-transformed and cancerous cells of human and mouse origin. By using proteomics, transcriptomics, and functional analysis, we assessed RNF144B's impact in cellular proliferation and transformation. Throughin vitroand in vivo experiments, we explored proliferation, DNA repair, cell cycle control, mitotic progression, and treatment resistance. Findings were contrasted with clinical datasets and bioinformatics analysis. Our research underscores RNF144B's pivotal role as a tumor suppressor, particularly in lung adenocarcinoma. In both human and mouse oncogene-expressing cells, RNF144B deficiency heightened cellular proliferation and transformation. Proteomic and transcriptomic analysis revealed RNF144B's novel function in mediating protein degradation associated with cell cycle progression, DNA damage response and genomic stability. RNF144B deficiency induced chromosomal instability, mitotic defects, and correlated with elevated aneuploidy and worse prognosis in human tumors. Furthermore, RNF144B-deficient lung adenocarcinoma cells exhibited resistance to cell cycle inhibitors that induce chromosomal instability. Supported by clinical data, our study suggests that RNF144B plays a pivotal role in maintaining genomic stability during tumor suppression.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

Abstract 6069: Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies

Abstract Background : Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade, which is critical for growth and survival of B-cell lymphoma or leukemia. BTK inhibitors have shown anti-tumor activity in patients with B-NHL. However, nearly half of the patients on current BTK inhibitors discontinue the treatment due to adverse effects and/or mutation-induced drug resistance. A degrader is a heterobifunctional chemical compound aimed at eliminating disease-causing proteins, rather than simply inhibiting proteins’ enzymatic function, through the ubiquitin-proteasome system (UPS)-mediated protein degradation. This approach offers a unique solution for addressing relapse or refractory disease by removing overexpressed and mutated proteins, unlike traditional inhibitors. We thus designed UBX-382, a degrader targeting BTK. Results : UBX-382 showed superior degradation activity for both wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration when compared to the competing degrader programs in diffuse large B-cell lymphoma cell line. UBX-382 effectively addressed 7 out of 8 previously reported resistant BTK mutants in in vitro experiments and outperformed ibrutinib, ARQ-531, and MT-802 in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. Following this, further optimization was carried out for potency and PK profile. We nominated the candidate for IND filing and are planning a phase 1 clinical trial. For the first-in-human, open-label, multicenter, phase 1 study, patients in part 1 (dose escalation with BOIN design) had R/R B-cell malignancies and had received at least 2 prior therapies. The candidate will be administered orally at 5 mg once daily followed by 2 days, for a 7-day schedule over 28-day cycles. Part 2 (dose expansion) consisted of disease-specific cohorts, including CLL/SLL, DLBCL, MCL, FL, MZL, and WM. The primary endpoints were safety and tolerability, along with the definition of the maximum tolerated dose. Secondary endpoints included pharmacokinetics/pharmacodynamics and preliminary efficacy. Conclusions : We have discovered potent BTK degrader exhibiting excellent anti-tumor activity in vitro and in vivo. In pre-clinical safety studies, the candidate of UBX demonstrated an acceptable safety profile. The candidate of UBX is currently preparing phase 1 clinical trials for hematological malignancies. Citation Format: Song Hee Lee, Sun-Mi Yoo, Ye Seul Lim, Han Wool Kim, Je Ho Ryu, Jong Hwan Lim. Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6069.

Upregulated Genes in Atrial Fibrillation Blood and the Left Atrium

Plain Language SummaryAtrial fibrillation (AF) is an aging disorder and risk factor for patients with cardiovascular disease, which is the leading cause of mortality among senior individuals. Despite intensive research, the prevalence of AF is rising in the senior population, and identifying new therapeutic targets and prognostic markers is therefore critically important. Our research focuses on the lysosomal pathway. Lysosome mediates protein degradation, which is activated in the heart and blood. We conducted bioinformatics analyses of publicly available databases to identify the upregulated genes and pathways in patients with AF. We analyzed gene expression data from blood and left atrium samples of patients with AF and sinus rhythm. We identified 12 upregulated genes in the blood and left atrium in patients with AF. These genes are mainly involved in lysosome-related functions. These findings suggest that the 12 genes identified in this study, especially lysosome-related genes, may be involved in the AF pathogenesis. Lysosome-related genes are important for cellular homeostasis and waste degradation. Therefore, their upregulation in patients with AF may play a role in disease development and progression. Our study proposes new insights into the underlying molecular mechanisms of AF and identifies potential biomarkers for predicting the AF onset risk or complications.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Integrated “-omics” analysis highlights the role of brassinosteroid signaling and antioxidant machinery underlying improved rice seed longevity during artificial aging treatment

Seed longevity is a critical characteristic in agriculture, yet the specific genes/proteins responsible for this trait and the molecular mechanisms underlying reduced longevity during seed aging remain largely elusive. Here we report the comparative proteome and metabolome profiling of three rice cultivars exhibiting varying degrees of aging tolerance: Dharial, an aging-tolerant cultivar; Ilmi, an aging-sensitive cultivar; and A2, a moderately aging-tolerant cultivar developed from the crossbreeding of Dharial and Ilmi. Artificial aging treatment (AAT) markedly reduced the germination percentage and enhanced the activities of antioxidant enzymes in all the cultivars. Further, proteomics results showed a key role of the ubiquitin (Ub)-proteasome pathway in the degradation of damaged proteins during AAT while other proteases were majorly reduced. In addition, proteins associated with energy production and protein synthesis were strongly reduced in Ilmi while these were majorly increased in A2 and Dharial. These, along with metabolomics results, suggest that Ub-proteasome mediated protein degradation during AAT results in the accumulation of free amino acids in Ilmi while tolerant cultivars potentially utilize those for energy production and synthesis of stress-related proteins, especially hsp20/alpha-crystallin family protein. Additionally, both Dharial and A2 seem to activate brassinosteroid signaling and suppress jasmonate signaling which initiates a signaling cascade that allows accumulation of enzymatic and non-enzymatic antioxidants for efficient detoxification of aging-induced ROS. Taken together, these results provide an in-depth understanding of the aging-induced changes in rice seeds and highlight key pathways responsible for maintaining seed longevity during AAT.

Paralogue-Specific Roles of SUMO1 and SUMO2/3 in Protein Quality Control and Associated Diseases.

Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single ubiquitin protein is expressed across eukaryotic organisms, multiple SUMO paralogues with distinct biomolecular properties have been identified in plants and vertebrates. Five SUMO paralogues have been characterized in humans, with SUMO1, SUMO2 and SUMO3 being the best studied. SUMO2 and SUMO3 share 97% protein sequence homology (and are thus referred to as SUMO2/3) but only 47% homology with SUMO1. To date, thousands of putative sumoylation substrates have been identified thanks to advanced proteomic techniques, but the identification of SUMO1- and SUMO2/3-specific modifications and their unique functions in physiology and pathology are not well understood. The SUMO2/3 paralogues play an important role in proteostasis, converging with ubiquitylation to mediate protein degradation. This function is achieved primarily through SUMO-targeted ubiquitin ligases (STUbLs), which preferentially bind and ubiquitylate poly-SUMO2/3 modified proteins. Effects of the SUMO1 paralogue on protein solubility and aggregation independent of STUbLs and proteasomal degradation have also been reported. Consistent with these functions, sumoylation is implicated in multiple human diseases associated with disturbed proteostasis, and a broad range of pathogenic proteins have been identified as SUMO1 and SUMO2/3 substrates. A better understanding of paralogue-specific functions of SUMO1 and SUMO2/3 in cellular protein quality control may therefore provide novel insights into disease pathogenesis and therapeutic innovation. This review summarizes current understandings of the roles of sumoylation in protein quality control and associated diseases, with a focus on the specific effects of SUMO1 and SUMO2/3 paralogues.

Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation.

Memory formation requires coordinated control of gene expression, protein synthesis, and ubiquitin-proteasome system (UPS)-mediated protein degradation. The catalytic component of the UPS, the 26S proteasome, contains a 20S catalytic core surrounded by two 19S regulatory caps, and phosphorylation of the 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Recently, RPT6 was also shown to act outside the proteasome where it has a transcription factor-like role in the hippocampus during memory formation. However, little is known about the proteasome-independent function of "free" RPT6 in the brain or during memory formation and whether phosphorylation of S120 is required for this transcriptional control function. Here, we used RNA-sequencing along with novel genetic approaches and biochemical, molecular, and behavioral assays to test the hypothesis that pRPT6-S120 functions independently of the proteasome to bind DNA and regulate gene expression during memory formation. RNA-sequencing following siRNA-mediated knockdown of free RPT6 revealed 46 gene targets in the dorsal hippocampus of male rats following fear conditioning, where RPT6 was involved in transcriptional activation and repression. Through CRISPR-dCas9-mediated artificial placement of RPT6 at a target gene, we found that RPT6 DNA binding alone may be important for altering gene expression following learning. Further, CRISPR-dCas13-mediated conversion of S120 to glycine on RPT6 revealed that phosphorylation at S120 is necessary for RPT6 to bind DNA and properly regulate transcription during memory formation. Together, we reveal a novel function for phosphorylation of RPT6 in controlling gene transcription during memory formation.

Abstract C137: Tumor-selective, chaperone-mediated targeting of ERK5 (MAPK7)

Abstract Extracellular signal-regulated kinase 5 (ERK5 or MAPK7) belongs to the mitogen-activated protein kinase family and has been associated with a wide range of cellular functions, including cell proliferation, migration, differentiation and survival. ERK5 has also been implicated in the onset and progression of several types of cancer. Importantly, studies comparing pharmacological inhibition versus genetic deletion or knockdown of ERK5 have revealed that it also possesses kinase activity-independent functions, suggesting that an ERK5 protein degrader approach may be required to completely block all ERK5-regulated signaling. To this end, we have explored applying Chaperone-mediated Protein Degradation (CHAMP) technology to target ERK5 protein. CHAMPs are heterobifunctional small molecules that mediate protein degradation by inducing proximity between a target protein and the HSP90 chaperone complex, resulting in target ubiquitination and degradation by the proteasome. Due to HSP90 being highly activated in cancer cells, CHAMPs also preferentially accumulate in tumors relative to normal tissues, resulting in an improved therapeutic index versus typical inhibitors. In order to chemically induce ERK5 degradation, CHAMP compounds were synthesized by covalently coupling ERK5- and HSP90-binding moieties through a short linker. CHAMP treatment of cancer cell lines resulted in formation of an ERK5-CHAMP-HSP90 ternary complex and subsequent proteasome-dependent ERK5 degradation. This in turn resulted in inhibition of ERK5 downstream signaling, leading to cell growth inhibition, cell cycle arrest and apoptosis in a panel of cancer cell lines. Further, in mouse xenograft models, CHAMPs displayed prolonged pharmacokinetics in tumors relative to plasma and normal tissues and strongly inhibited tumor growth at tolerated doses. Based on these findings, ERK5-targeted CHAMPs represents a promising new approach to disrupt the entire function of ERK5 for the of treatment cancer. Citation Format: Kevin P Foley, Xiangcai Yang, Wei Yin, Mengmeng Xu, Mingkai Wang, Qinglin Ding, Yan Dai, Haoxin Zhou, Ignazia Tusa, Alessio Menconi, Zoe Lombardi, Alessandro Tubita, Thomas L Prince, Guoqiang Wang, Elisabetta Rovida, Weiwen Ying. Tumor-selective, chaperone-mediated targeting of ERK5 (MAPK7) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C137.

USP12 promotes nonsmall cell lung cancer progression through deubiquitinating and stabilizing RRM2.

RRM2 is the catalytic subunit of ribonucleotide reductase (RNR), which catalyzes de novo synthesis of deoxyribonucleotide triphosphates (dNTPs) and plays critical roles in cancer cell proliferation. RRM2 protein level is controlled by ubiquitination mediated protein degradation system; however, its deubiquitinase has not been identified yet. Here we showed that ubiquitin-specific peptidase 12 (USP12) directly interacts with and deubiquitinates RRM2 in non-small cell lung cancer (NSCLC) cells. Knockdown of USP12 causes DNA replication stress and retards tumor growth in vivo and in vitro. Meanwhile, USP12 protein levels were positively correlated to RRM2 protein levels in human NSCLC tissues. In addition, high expression of USP12 was associated with poor prognosis in NSCLC patients. Therefore, our study reveals that USP12 is a RRM2 regulator and targeting USP12 could be considered as a potential therapeutical strategy for NSCLC treatment.

Overexpression of ARM repeat/U-box containing E3 ligase, PUB2 positively regulates growth and oxidative stress response in Arabidopsis.

Plant growth and development are governed by selective protein synthesis and degradation. Ubiquitination mediated protein degradation is governed by activating enzyme E1 followed by conjugating enzyme E2 and E3 ligase. Plant Armadillo (ARM) repeat/U-box (PUB) protein family is one of the important classes of E3 ligase. We studied the function of AtPUB2 by loss-of-function (knockout and knock down mutants) and gain-of-function (CaMV 35S promoter driven overexpression lines) approach in Arabidopsis. Under normal growth condition, we observed that loss-of-function mutant plants did not show any significant difference in growth when compared with wild-type possibly due to functional redundancy between PUB2 and PUB4. However, AtPUB2-OE lines exhibit early flowering and improved vegetative growth. Also, AtPUB2-OE seedlings showed sensitive phenotype in the presence of exogenous cytokinin. We found that AtPUB2 expression is induced under oxidative stress. Subcellular localization analysis shows that AtPUB2 is predominantly localized in the nucleus. We performed the phenotypic analysis under oxidative stress condition induced by methyl viologen (MV) and observed that overexpression lines display tolerance to oxidative stress in light and dark conditions. Furthermore, we found less amount of ROS accumulation, enhanced proline accumulation and decreased levels of MDA after MV treatment in AtPUB2-OE lines. PUB2-OE lines showed enhanced oxidative stress marker genes expression. By in vitro auto-ubiquitination assay, we also show that it possesses the E3 ligase activity. Overall, our findings suggest the possible role of AtPUB2 in plants ability to tolerate oxidative stress by enhancing the activity of antioxidant enzymes, which in turn improves ROS scavenging activity and homeostasis.

Multimodal Wnt signalling in the mouse neocortex

The neocortex is the site of higher cognitive functions and its development is tightly regulated by cell signalling pathways. Wnt signalling is inexorably linked with neocortex development but its precise role remains unclear. Most studies demonstrate that Wnt/β-catenin regulates neural progenitor self-renewal but others suggest it can also promote differentiation. Wnt/STOP signalling is a novel branch of the Wnt pathway that stabilizes proteins during G2/M by inhibiting glycogen synthase kinase 3 (GSK3)-mediated protein degradation. Recent data from our work in Da Silva et al. (2021) demonstrate that Wnt/STOP is involved in neocortex development where, by stabilizing the neurogenic transcription factors Sox4 and Sox11, it promotes neural progenitor differentiation. We also show that Wnt/STOP regulates asymmetric cell division and cell cycle dynamics in apical and basal progenitors, respectively. Our study reveals a division of labour in the Wnt signalling pathway by suggesting that Wnt/STOP is the primary driver of cortical neurogenesis while Wnt/β-catenin is mainly responsible for self-renewal. These results resolve a decades-old question on the role of Wnt signalling in cortical neural progenitors.

The In-Cell Western immunofluorescence assay to monitor PROTAC mediated protein degradation.

The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.

CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.

Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfill well-defined roles in protein folding and conformational stability via ATP-dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23, and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone-mediated folding process. However, chaperones are also involved in proteasomal and lysosomal degradation of client proteins. Like folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C-terminal Hsp70-binding protein (CHIP/STUB1). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome and autophagosome-lysosome systems. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation.

Targeted Protein Degradation for Infectious Diseases: from Basic Biology to Drug Discovery.

Targeted protein degradation (TPD) is emerging as one of the most innovative strategies to tackle infectious diseases. Particularly, proteolysis-targeting chimera (PROTAC)-mediated protein degradation may offer several benefits over classical anti-infective small-molecule drugs. Because of their peculiar and catalytic mechanism of action, anti-infective PROTACs might be advantageous in terms of efficacy, toxicity, and selectivity. Importantly, PROTACs may also overcome the emergence of antimicrobial resistance. Furthermore, anti-infective PROTACs might have the potential to (i) modulate "undruggable" targets, (ii) "recycle" inhibitors from classical drug discovery approaches, and (iii) open new scenarios for combination therapies. Here, we try to address these points by discussing selected case studies of antiviral PROTACs and the first-in-class antibacterial PROTACs. Finally, we discuss how the field of PROTAC-mediated TPD might be exploited in parasitic diseases. Since no antiparasitic PROTAC has been reported yet, we also describe the parasite proteasome system. While in its infancy and with many challenges ahead, we hope that PROTAC-mediated protein degradation for infectious diseases may lead to the development of next-generation anti-infective drugs.

GSK-3β phosphorylation of DHX33 leads to its ubiquitination mediated protein degradation

DHX33 is a member of DEAD/H box protein family, and is involved in both RNA and DNA metabolism. It plays diverse roles in multiple cellular activities. DHX33 overexpression has been found to promote the development of many human cancers. However, the underlying mechanism to explain its high expression in cancer cells remains incompletely resolved. In this study, with both human cancer cell lines and normal fibroblasts, we found glycogen synthase kinase 3β (GSK-3β) regulates DHX33 protein stability. This is through its direct phosphorylation of DHX33 on T482, which triggers ubiquitination mediate protein degradation. We further identified one of the major ubiquitination sites of DHX33 to be on its N-terminal K94, a critical residue previously found to be important and highly conserved for ATP binding and helicase activity. Our study for the first time reveals an important upstream regulator, GSK-3β, as a critical kinase to phosphorylate DHX33 at the post-translational level leading to its degradation. Moreover, cancer cells have frequent GSK3β deactivation to disrupt this signaling cascade. Therefore, DHX33 is stabilized in many cancer cells as compared to normal cells. Our study unveils an important post-translational regulation of DHX33 in cells and further unveils a novel mechanism for DHX33 overexpression in cancer cells.

Global ubiquitinome analysis reveals the role of E3 ubiquitin ligase FaBRIZ in strawberry fruit ripening.

Ubiquitination is an important post-translational modification that mediates protein degradation in eukaryotic cells, participating in multiple biological processes. However, the profiling of protein ubiquitination and the function of this crucial modification in fruit ripening remain largely unknown. In this study, we found that suppression of proteasome by the inhibitor MG132 retarded strawberry fruit ripening. Using K-ɛ-GG antibody enrichment combined with high-resolution mass spectrometry, we performed a comprehensive ubiquitinome analysis in strawberry fruit. We identified 2947 ubiquitination sites for 2878 peptides within 1487 proteins, which are involved in a variety of cellular functions. The lysine at position 48 (K48)-linked poly-ubiquitin chains appeared to be the most prevalent type of modification among the identified ubiquitinated proteins. A large number of ubiquitination sites exhibited altered ubiquitination levels after proteasome inhibition, including those within ripening-related proteins associated with sugar and acid metabolism, cell wall metabolism, anthocyanin synthesis, and ABA biosynthesis and signalling. We further demonstrated that FaBRIZ, a RING-type E3 ligase, functions as a negative regulator of ripening in strawberry fruit. Our findings highlight the critical regulatory roles of protein ubiquitination in fruit ripening. The ubiquitinome data provide a basis for further exploration of the function of ubiquitination on specific proteins.

The Key Role of Lysosomal Protease Cathepsins in Viral Infections.

Cathepsins encompass a family of lysosomal proteases that mediate protein degradation and turnover. Although mainly localized in the endolysosomal compartment, cathepsins are also found in the cytoplasm, nucleus, and extracellular space, where they are involved in cell signaling, extracellular matrix assembly/disassembly, and protein processing and trafficking through the plasma and nuclear membrane and between intracellular organelles. Ubiquitously expressed in the body, cathepsins play regulatory roles in a wide range of physiological processes including coagulation, hormone secretion, immune responses, and others. A dysregulation of cathepsin expression and/or activity has been associated with many human diseases, including cancer, diabetes, obesity, cardiovascular and inflammatory diseases, kidney dysfunctions, and neurodegenerative disorders, as well as infectious diseases. In viral infections, cathepsins may promote (1) activation of the viral attachment glycoproteins and entry of the virus into target cells; (2) antigen processing and presentation, enabling the virus to replicate in infected cells; (3) up-regulation and processing of heparanase that facilitates the release of viral progeny and the spread of infection; and (4) activation of cell death that may either favor viral clearance or assist viral propagation. In this review, we report the most relevant findings on the molecular mechanisms underlying cathepsin involvement in viral infection physiopathology, and we discuss the potential of cathepsin inhibitors for therapeutical applications in viral infectious diseases.

N7 -SSPP fusion gene improves salt stress tolerance in transgenic Arabidopsis and soybean through ROS scavenging.

Considerable signal crosstalk exists in the regulatory network of senescence and stress response. Numerous senescence-associated genes are also involved in plant stress tolerance. However, the underlying mechanisms and application potential of these genes in stress-tolerant crop breeding remain poorly explored. We found that overexpression of SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), a negative regulator of leaf senescence, significantly improved plant salt tolerance by increasing reactive oxygen species (ROS) scavenging in both Arabidopsis and soybean. However, overexpression of SSPP severely suppressed normal plant growth, limiting its direct use in agriculture. We previously revealed that the N-terminal 1-14 residues of ACS7 (termed 'N7 ') negatively regulated its protein stability through the ubiquitin/proteasome pathway, and the N7 -mediated protein degradation was suppressed by environmental and senescence signals. To avoid the adverse effects of SSPP, the N7 element was fused to the N-terminus of SSPP. We demonstrated that N7 -SSPP fusion gene effectively rescued SSPP-induced growth suppression but maintained enhanced salt tolerance in Arabidopsis and soybean. Particularly, N7 -SSPP enhanced tolerance to long-term salt stress and increased seed yield in soybean. These results suggest that N7 -SSPP overcomes the disadvantages of SSPP on plant growth inhibition and effectively improves salt tolerance through enhanced ROS scavenging, providing an effective strategy of using posttranslational regulatory element for salt-tolerant crop breeding.

Enriching Proteolysis Targeting Chimeras with a Second Modality: When Two Are Better Than One.

Proteolysis targeting chimera (PROTAC)-mediated protein degradation has prompted a radical rethink and is at a crucial stage in driving a drug discovery transition. To fully harness the potential of this technology, a growing paradigm toward enriching PROTACs with other therapeutic modalities has been proposed. Could researchers successfully combine two modalities to yield multifunctional PROTACs with an expanded profile? In this Perspective, we try to answer this question. We discuss how this possibility encompasses different approaches, leading to multitarget PROTACs, light-controllable PROTACs, PROTAC conjugates, and macrocycle- and oligonucleotide-based PROTACs. This possibility promises to further enhance PROTAC efficacy and selectivity, minimize side effects, and hit undruggable targets. While PROTACs have reached the clinical investigation stage, additional steps must be taken toward the translational development of multifunctional PROTACs. A deeper and detailed understanding of the most critical challenges is required to fully exploit these opportunities and decisively enrich the PROTAC toolbox.