Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

The role of 5′-adenosine monophosphate-activated protein kinase(AMPK)in skeletal muscle atrophy

As a key coordinator of metabolism,AMP-activated protein kinase(AMPK)is vitally involved in skeletal muscle maintenance.AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle.AMPK is activated by phosphorylation and exerts its function as a kinase in many processes,including synthesis and degradation of proteins,mitochondrial biogenesis,glucose uptake,and fatty acid and cholesterol metabolism.Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass.The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation.In this review,we discuss the role of AMPK in skeletal muscle metabolism and atrophy.We also discuss targeting AMPK for skeletal muscle treatment,including exercise,AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin,and low-level lasers.These studies show the important roles of AMPK in regulating muscle metabolism and function;thus,the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.

Fangchinoline induces antiviral response by suppressing STING degradation

The stimulator of interferon genes(STING),an integral adaptor protein in the DNA-sensing pathway,plays a pivotal role in the innate immune response against infections.Additionally,it presents a valuable therapeutic target for infectious diseases and cancer.We observed that fangchinoline(Fan),a bis-benzylisoquinoline alkaloid(BBA),effectively impedes the replication of vesicular stomatitis virus(VSV),encephalomyocarditis virus(EMCV),influenza A virus(H1N1),and herpes simplex virus-1(HSV-1)in vitro.Fan treatment significantly reduced the viral load,attenuated tissue inflammation,and improved survival in a viral sepsis mouse model.Mechanistically,Fan activates the antiviral response in a STING-dependent manner,leading to increased expression of interferon(IFN)and interferon-stimulated genes(ISGs)for potent antiviral effects in vivo and in vitro.Notably,Fan interacts with STING,preventing its degradation and thereby extending the activation of IFN-based antiviral responses.Collectively,our findings highlight the potential of Fan,which elicits antiviral immunity by suppressing STING degradation,as a promising candidate for antiviral therapy.

Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

Background： The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1,6- and 26-d-old pigs were studied during 1） euinsulinemic-euglycemic-euaminoacidemic, 2） euinsulinemic-euglycemiohyperaminoacidemic, and 3） hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1） euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2） euinsulinemic-euglycemic-hypoaminoacidemic- hyperleucinemic, and 3） euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 （MAFbx） and muscle RING-finger protein-1 （MuRF1） and autophagy-lysosome systems, i.e., unc51-1ike kinase 1 （UKL1）, microtubule-associated protein light chain 3 （LC3）, and lysosomal-associated membrane protein 2 （Lamp-2）. For comparison, we measured ribosomal protein 56 （rpS6） and eukaryotic initiation factor 4E （elF4E） activation, components of translation initiation. Results： Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-11/LC3-1 ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of elF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of elF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development.

ATP regulates the phosphorylation and degradation of myofibrillar proteins in ground ovine muscle

Phosphorylation post-translational modification plays an important role in postmortem muscle quality traits. Adenosine triphosphate(ATP) is an energy source and a key substrate of phosphorylation which provides the phosphatase groups to proteins in the presence of protein kinases. However, in postmortem muscle, the effects of ATP content on phosphorylation are poorly studied. The study investigated the effect of ATP on protein phosphorylation and degradation in postmortem ovine muscle. The ground muscle with/without additional ATP were treated/control groups and stored at 25 and 4℃, respectively. The ATP content led to different changes of p H value between the ATP-treated and control groups. The phosphorylation level of myofibrillar proteins was higher(P<0.05) in ATP-treated group compared to the control group at both temperatures, which suggested that ATP played a vital role in postmortem protein phosphorylation. A slower degradation rate of μ-calpain, desmin and troponin T was observed in the ATP-treated group which showed that there was a negative relationship between ATP level and the degradation of proteins. These observations clearly highlighted the role of ATP on the development of meat quality by regulating the phosphorylation and degradation of myofibrillar proteins in postmortem ovine muscle.

The role of sequestosome 1/p62 protein in amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.

Isolation,Identification of Bacillus Thuringiensis/Cereus and Its Enhancement on Protein Wastewater Treatment by Rhodobacter Sphaeroides

In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein removal of R.sphaeroides were investigated.Results showed that strain L2,being identified as Bacillus thuringiensis/cereus,had a high potential for producing protease with a production of 295 U/m L.The optimal B.thuringiensis/cereus(40 μL) could significantly increase protein degradation of R.sphaeroides.Protein removal and biomass production were improved by 483% and 67%,respectively.R.sphaeroides/total biomass production was more than 95%.Theoretical analysis revealed that R.sphaeroides syntrophically interacted with B.thuringiensis/cereus.Protein degradation of B.thuringiensis/cereus provided small molecule substrates(VFAs) for R.sphaeroides growth and cells materials synthesis.

Issues with Tropical and Temperate Ensilage Protein and Amino Acid Feeds Utilization: A Research Note

Alfalfa protein breakdown was to soluble NPN of oligopeptide-N, AA-N, amide-N, amine-N and NH3-N. Acidity (pH) and moisture (Aw) are critical in determining extent of fermentation and changes in composition. Further changes in digestive flows and post-prandial plasma AA are indicators of protein status. Dual-purpose cropping and tree plant cropping was with ensiling management of the undergrowth. On-farm field-drying and probiotic additives are promising. It is suggested acidity with propionic acid and microbial inoculants together with field-drying and chop length are required to optimize profile qualities in silage. It is proposed use of denaturing with acid and dust cropping with a hypothetical PNA-Auxin repressor to plant protease. Further study with field-drying to follow is needed. Feeding HIS, ARG and LEU AA supplement to change GRH and GH profiles could be used to promote LBM in production. Dual-purpose cropping can expand subsistence to mixed farming with expanded livestock products and services and resources. PNA-Auxin and PNA-ARF penetrates the plant shoot tips to deliver a TF mRNA to boost proteins in residual cell tissues. Ensiled % AA-N delivery per os to per duodenum was higher;yet total AA-N flow was higher in the control. It is suggested that “bulk” flow was less but with a “tighter” conversion on TAA. FAA was 145% higher in the ensiled versus the fresh control indicating the ENU with less PFAA supplied. FAA on the ensiled diet is high inferred to be more soluble and escape lower from the rumen. WSC are less supplied in fermented forage with VFA being lower and presenting the question whether WSC should be supplied for energy and also with EFE through breaking down of polymers of lignocellulose. It was surmised, although not known, that higher dilution rate (% hr-1) was true on the fresh diet compared to the ensiled although end-products may initially detract with feed but that further digestion in the fresh feed may be higher with intake. Plasma AA before and after absorption or feeding are indicators of synthesis and breakdown. No data was available on N status;protein nutrition on neat silage was probably due to net efflux of AA with mobilization before influx with feeding and subsequent insulin action for uptake. Estuarine aquatic plant spp., water hyacinth used in the Philippines and duckweed studies in Australia, and post-harvest treatment with chemical additives and anti-microbial agents to help control potential transfer of diseases. “Greens” as supplements has yet to be established for anti-microbial properties for animal health and welfare. In conclusion, alfalfa silage fed at standard 0.6 cm particle size and wilted led to dramatic changes with AA breakdown, dramatic changes in duodenal AA flows from escape and recapture into microbial cells. Also N status of animals was compromised by lack of adequate “stores”, mobilized, resulting in a net decrease in total plasma AA with insulin-dependent uptake to tissue.

2007 International Symposium on Protein Modification and Degradation in Beijing(SPMDB2007)

Presidents of Symposium Depei Liu,President of Chinese Academy of Medical Sciences Zhu Chen,Vice President of Chinese Academy ofSciences.

Degradation of Cry1Ab Protein Within Transgenic Bt Maize Tissue by Composite Microbial System of MC1

Environmental safety issues involved in transgenic plants have become the concern of researchers, practitioners and policy makers in recent years. Potential differences between Bt maize（ND1324 and ND2353 expressing the insecticidal Cry1Ab protein） and near-isogenic non-Bt varieties（ND1392 and ND223） in their influence on the composite microbial system of MC1 during the fermentation process were studied during 2011-2012. Cry1Ab protein in Bt maize residues didn＇t affect characteristics of lignocellulose degradation by MC1, pH of fermentation broth decreasing at initial stage and increasing at later stage of degradation. The quality of various volatile products in fermentation broth showed that no signifi cant difference of residues fermentation existed between Bt maize and non-Bt maize. During the fermentation MC1 efficiently degraded maize residues by 83%-88%, and cellulose, hemicelluloses and lignin content decreased by 70%-72%, 72%-75% and 30%-37%, respectively. Besides that, no consistent difference was found between Bt and non-Bt maize residues lignocellulose degradation by MC1 during the fermentation process. MC1 degraded 88%-89% Cry1Ab protein in Bt maize residues, and in the fermentation broth of MC1 and bacteria of MC1 Cry1Ab protein was not detected. DGGE profi le analyses revealed that the microbial community drastically changed during 1-3 days and became stable until the 9th day. Though the dominant strains at different fermentation stages had signifi cantly changed, no difference on the dominant strains was observed between Bt and non-Bt maize at different stages. Our study indicated that Cry1Ab protein did not infl uence the growth characteristic of MC1.

Winery by‑products as a feed source with functional properties:dose–response effect of grape pomace,grape seed meal,and grape seed extract on rumen microbial community and their fermentation activity in RUSITEC

Background Grape and winery by-products have nutritional values for cattle and also contain functional compounds like phenols,which not only bind to protein but can also directly affect microbiota and their function in the rumen.We characterized the nutritional and functional effects of grape seed meal and grape pomace as well as an effective dosage of grape phenols on ruminal microbiota and fermentation characteristics using a rumen simulation technique.Results Six diets(each n=8)were compared including a control diet(CON,no by-product),a positive control diet(EXT,CON+3.7%grape seed extract on a dry matter(DM)basis),two diets with grape seed meal at 5%(GS-low)and 10%(GS-high),and two diets with grape pomace:at 10%(GP-low)and 20%(GP-high),on a DM basis.The inclusion of the by-product supplied total phenols at 3.4%,0.7%,1.4%,1.3%,and 2.7%of diet DM for EXT,GS-low,GS-high,GP-low,and GP-high,respectively.Diets were tested in four experimental runs.All treatments decreased ammonia concentrations and the disappearances of DM and OM(P<0.05)compared to CON.EXT and GP-high lowered butyrate and odd-and branch-chain short-chain fatty acids while increased acetate compared to CON(P<0.05).Treatments did not affect methane formation.EXT decreased the abundance of many bacterial genera including those belonging to the core microbiota.GP-high and EXT consistently decreased Olsenella and Anaerotipes while increased Ruminobacter abundances.Conclusion The data suggest that the inclusion of winery by-products or grape seed extract could be an option for reducing excessive ammonia production.Exposure to grape phenols at a high dosage in an extract form can alter the rumen microbial community.This,however,does not necessarily alter the effect of grape phenols on the microbial community function compared to feeding high levels of winery by-products.This suggests the dominant role of dosage over the form or source of the grape phenols in affecting ruminal microbial activity.In conclusion,supplementing grape phenols at about 3%of diet DM is an effective dosage tolerable to ruminal microbiota.

Ciclopirox inhibits SARS-CoV-2 replication by promoting the degradation of the nucleocapsid protein

The nucleocapsid protein(NP)plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life.Despite its vital role in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)assembly and host inflammatory response,it remains an unexplored target for drug development.In this study,we identified a small-molecule compound(ciclopirox)that promotes NP degradation using an FDA-approved library and a drug-screening cell model.Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation.Ciclopirox induced abnormal NP aggregation through indirect interaction,leading to the formation of condensates with higher viscosity and lower mobility.These condensates were subsequently degraded via the autophagy-lysosomal pathway,ultimately resulting in a shortened NP half-life and reduced NP expression.Our results suggest that NP is a potential drug target,and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.

Expanding the horizons of targeted protein degradation:A non-small molecule perspective

Targeted protein degradation(TPD)represented by proteolysis targeting chimeras(PROTACs)marks a significant stride in drug discovery.A plethora of innovative technologies inspired by PROTAC have not only revolutionized the landscape of TPD but have the potential to unlock functionalities beyond degradation.Non-small-molecule-based approaches play an irreplaceable role in this field.A wide variety of agents spanning a broad chemical spectrum,including peptides,nucleic acids,antibodies,and even vaccines,which not only prove instrumental in overcoming the constraints of conventional small molecule entities but also provided rapidly renewing paradigms.Herein we summarize the burgeoning non-small molecule technological platforms inspired by PROTACs,including three major trajectories,to provide insights for the design strategies based on novel paradigms.

Hydrophobic tag tethering degrader as a promising paradigm of protein degradation:Past,present and future perspectives

Small molecule inhibitors have dominated the pharmaceutical landscape for a long time as the primary therapeutic paradigm targeting pathogenic proteins.However,their efficacy heavily relies on the amino acid composition and spatial constitution of proteins,rendering them susceptible to drug resistance and failing to target undruggable proteins.In recent years,the advent of targeted protein degradation(TPD)technology has captured substantial attention from both industry and academia.Employing an event-driven mode,TPD offers a novel approach to eliminate pathogenic proteins by promoting their degrada-tion,thus circumventing the limitations associated with traditional small molecule inhibitors.Hydropho-bic tag tethering degrader(HyTTD)technology represents one such TPD approach that is currently in the burgeoning stage.HyTTDs employ endogenous protein degradation systems to induce the degrada-tion of target proteins through the proteasome pathway,which displays significant potential for medical value.In this review,we provide a comprehensive overview of the development history and the reported mechanism of action of HyTTDs.Additionally,we delve into the physiological roles,structure-activity re-lationships,and medical implications of HyTTDs targeting various disease-associated proteins.Moreover,we propose insights into the challenges that necessitate resolution for the successful development of HyTTDs,with the ultimate goal of initiating a new age of clinical treatment leveraging the immense po-tential of HyTTDs.

Artificial Nucleic Acid Tractor-Directed Simultaneous Depletion of Oncogenic Membrane Proteins Without Hijacking Proteolysis-Specific Actuator

Targeted protein degradation(TPD)is an emerging tool for degrading proteins of interest,which affords an attractive modality for cancer therapy.However,the present TPD technologies must engage a proteolysis-specific actuator to initiate degradation of targeted proteins in the proteasome or lysosome.Herein,we report an artificial tractor that can induce endocytosis-mediated protein depletion without hijacking a proteolysis-specific actuator.In this design,bispecific aptamer chimeras(BSACs)are established,which can bridge human epidermal growth factor receptor 2(ErbB-2),an important biomarker in a common important biomarker in cancer,with membrane proteins of interest.Taking advantage of the property of aptamer-induced endocytosis and digestion of ErbB-2,another membrane protein is translocated into the lysosome in a hitchhike-like manner,resulting in lysosomal proteolysis along with ErbB-2.This strategy frees the TPD from the fundamental limitation of proteolysis-specific actuator and allows simultaneous regulation of the quantity and function of two oncogenic receptors in a cell-type-specific manner,expanding the application scope of TPD-based therapeutics.

WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.

Concomitant NH_4^+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.

The expression of Usp26 gene in mouse testis and brain

Deubiquitinating enzymes （DUBs） play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 （USP26） is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription （RT）-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids （steps 9-16）, Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial ceils, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

CLONING AND CHARACTERIZATION OF HUMANUBIQUITIN BINDING ENZYME 2 cDNA

To clone and identify the gene encoding human ubiquitin binding enzym e 2 and study its expression pattern. Methods. According to the sequence of human EST, which is highly homologous to t he mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformati cs technique and its expression pattern was studied by using multiple tissue No rthern blot. Results. Two cDNA clones encoding human ubiquitin conjugating enzyme have been i solated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows th at they are expressed exclusively in adult human heart, placenta, and pancreas b ut no transcripts can be detected in brain, lung, liver, skeletal muscle or kidn ey. Conclusions. The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin con jugating enzymes play a central role in the expression regulation on the level o f post translation.

Targeted Degradation of DNA/RNA Binding Proteins via Covalent Hydrophobic Tagging

Targeted protein degradation(TPD)holds great promise for biological inquiry and therapeutic development.However,it still remains elusive to destruct DNA/RNA binding proteins(DBPs/RBPs)previously deemed undruggable.Herein,we report ligandassisted covalent hydrophobic tagging(LACHT)as a modular strategy for TPD of these difficult-totarget proteins.Guided by a noncovalent protein ligand,LACHT leverages a reactive N-acyl-N-alkyl sulfonamide group to covalently label the protein target with a hydrophobic adamantane,which further engages the cellular quality control mechanism to induce proteolytic degradation.Using a smallmolecule ligand,we demonstrated that LACHT allowed TPD of a DBP,bromodomain-containing protein 4,in human leukemia cells with high efficiency.Mechanistic studies revealed that LACHT-mediated TPD dependent on ligand-directed irreversible tagging and the covalently labeled proteins underwent polyubiquitination before removal through both the proteasome and the lysosome.Furthermore,when an RNA ligand was employed,we showed that LACHT also enabled TPD of an RBP,Lin28a,leading to upregulation of its downstream let-7 miRNA.This study thus provides a generalizable platform to expand the TPD toolbox for biomedical applications.