Control Of Protein Degradation Research Articles

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

MicroRNA-Triggered Programmable DNA-Encoded Pre-PROTACs for Cell-Selective and Controlled Protein Degradation.

Proteolysis-targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on-demand protein degradation. Here, we developed a miRNA-initiated assembled pre-PROTAC (miRiaTAC) platform that enables the on-demand activation and termination of target degradation in a cell type-specific manner. Using miRNA-21 as a model, we engineered DNA hairpins labeled with JQ-1 and pomalidomide and facilitated the modular assembly of DNA-encoded pre-PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR-21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre-PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre-PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near-infrared light-mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

MicroRNA‐Triggered Programmable DNA‐Encoded Pre‐PROTACs for Cell‐Selective and Controlled Protein Degradation

Proteolysis‐targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on‐demand protein degradation. Here, we developed a miRNA‐initiated assembled pre‐PROTAC (miRiaTAC) platform that enables the on‐demand activation and termination of target degradation in a cell type‐specific manner. Using miRNA‐21 as a model, we engineered DNA hairpins labeled with JQ‐1 and pomalidomide and facilitated the modular assembly of DNA‐encoded pre‐PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR‐21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre‐PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre‐PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near‐infrared light‐mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

ProSAAS is preferentially up-regulated during homeostatic scaling and reduces amyloid plaque burden in the 5xFAD mouse hippocampus.

The accumulation of β-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha-synuclein and β-amyloid fibrillation invitro and is up-regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.

Dysregulation of ceramide metabolism causes phytoceramide-dependent induction of the unfolded protein response.

The unfolded protein response (UPR) detects and mitigates the harmful effects of dysregulated endoplasmic reticulum (ER) function. The UPR has been best characterized as a protein quality control response, and the sole UPR sensor in yeast, Ire1, is known to detect misfolded ER proteins. However, recent work suggests the UPR can also sense diverse defects within the ER membrane, including increased fatty acid saturation and altered phospholipid abundance. These and other lipid-related stimuli have been referred to as lipid bilayer stress and may be sensed independently through Ire1's transmembrane domain. Here, we show that the loss of Isc1, a phospholipase that catabolizes complex ceramides, causes UPR induction, even in the absence of exogenous stress. A series of chemical and genetic approaches identified a requirement for very long-chain fatty acid (VLCFA)-containing phytoceramides for UPR induction. In parallel, comprehensive lipidomics analyses identified large increases in the abundance of specific VLCFA-containing phytoceramides in the isc1Δ mutant. We failed to identify evidence of an accompanying defect in protein quality control or ER-associated protein degradation. These results extend our understanding of lipid bilayer stress in the UPR and provide a foundation for mechanistic investigation of this fascinating intersection between ceramide metabolism, membrane homeostasis, and the UPR.

Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis.

Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.

Ultrasound Controllable Release of Proteolysis Targeting Chimeras for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, which is highly aggressive and incurable. Here, we proposed an ultrasound activatable bromodomain-containing protein 4 (BRD4) proteolysis targeting chimera (PROTAC) release strategy for the first time for precisely controlled protein degradation in preclinical TNBC model. Through combination of PROTAC and ultrasound-targeted microbubble destruction (UTMD) technology, the present strategy also aims to concurrently solve the major limitations of poor loading capacity of microbubbles and undesirable targeting and membrane permeability of PROTAC. PROTAC (ARV-825)-encapsulated microbubbles, ARV-MBs, were developed for the efficacious treatment of TNBC invitro and invivo. The microbubbles we synthesized showed ultrasound-responsive drug release ability, which could effectively promote the penetration of PROTAC into tumor site and tumor cell. Under ultrasound, ARV-MBs could play an effective antitumor effect by potentiating the ubiquitination and degradation of BRD4 in tumor. The current study may provide a new idea for promoting clinical translation of drug-loaded microbubbles and PROTAC, and offer a new efficacious therapeutic modality for TNBC.

USP38 exacerbates pressure overload-induced left ventricular electrical remodeling

BackgroundUbiquitin-specific protease 38 (USP38), belonging to the USP family, is recognized for its role in controlling protein degradation and diverse biological processes. Ventricular arrhythmias (VAs) following heart failure (HF) are closely linked to ventricular electrical remodeling, yet the specific mechanisms underlying VAs in HF remain inadequately explored. In this study, we examined the impact of USP38 on VAs in pressure overload-induced HF.MethodsCardiac-specific USP38 knockout mice, cardiac-specific USP38 transgenic mice and their matched control littermates developed HF induced by aortic banding (AB) surgery. After subjecting the mice to AB surgery for a duration of four weeks, comprehensive investigations were conducted, including pathological analysis and electrophysiological assessments, along with molecular analyses.ResultsWe observed increased USP38 expression in the left ventricle of mice with HF. Electrocardiogram showed that the USP38 knockout shortened the QRS interval and QTc, while USP38 overexpression prolonged these parameters. USP38 knockout decreased the susceptibility of VAs by shortening action potential duration (APD) and prolonging effective refractory period (ERP). In addition, USP38 knockout increased ion channel and Cx43 expression in ventricle. On the contrary, the increased susceptibility of VAs and the decreased expression of ventricular ion channels and Cx43 were observed with USP38 overexpression. In both in vivo and in vitro experiments, USP38 knockout inhibited TBK1/AKT/CAMKII signaling, whereas USP38 overexpression activated this pathway.ConclusionOur data indicates that USP38 increases susceptibility to VAs after HF through TBK1/AKT/CAMKII signaling pathway, Consequently, USP38 may emerge as a promising therapeutic target for managing VAs following HF.

A BRD4‐Targeting Photothermal Agent for Controlled Protein Degradation

AbstractBRD4 protein plays a pivotal role in cell cycle regulation and differentiation. Disrupting the activity of BRD4 has emerged as a promising strategy for inhibiting the growth and proliferation of cancer cells. Herein, we introduced a BRD4‐targeting photothermal agent for controlled protein degradation, aiming to enhance low‐temperature photothermal therapy (PTT) for cancer treatment. By incorporating a BRD4 protein inhibitor into a cyanine dye scaffold, the photothermal agent specifically bond to the bromodomain of BRD4. Upon low power density laser irradiation, the agent induced protein degradation, directly destroying the BRD4 structure and inhibiting its transcriptional regulatory function. This strategy not only prolonged the retention time of the photothermal agent in cancer cells but also confined the targeted protein degradation process solely to the tumor tissue, minimizing side effects on normal tissues through the aid of exogenous signals. This work established a simple and feasible platform for future PTT agent design in clinical cancer treatment.

A BRD4-Targeting Photothermal Agent for Controlled Protein Degradation.

BRD4 protein plays a pivotal role in cell cycle regulation and differentiation. Disrupting the activity of BRD4 has emerged as a promising strategy for inhibiting the growth and proliferation of cancer cells. Herein, we introduced a BRD4-targeting photothermal agent for controlled protein degradation, aiming to enhance low-temperature photothermal therapy (PTT) for cancer treatment. By incorporating a BRD4 protein inhibitor into a cyanine dye scaffold, the photothermal agent specifically bond to the bromodomain of BRD4. Upon low power density laser irradiation, the agent induced protein degradation, directly destroying the BRD4 structure and inhibiting its transcriptional regulatory function. This strategy not only prolonged the retention time of the photothermal agent in cancer cells but also confined the targeted protein degradation process solely to the tumor tissue, minimizing side effects on normal tissues through the aid of exogenous signals. This work established a simple and feasible platform for future PTT agent design in clinical cancer treatment.

The DNA repair kinase ATM regulates CD13 expression and cell migration.

Classically, ATM is known for its role in sensing double-strand DNA breaks, and subsequently signaling for their repair. Non-canonical roles of ATM include transcriptional silencing, ferroptosis, autophagy and angiogenesis. Angiogenesis mediated by ATM signaling has been shown to be VEGF-independent via p38 signaling. Independently, p38 signaling has been shown to upregulate metalloproteinase expression, including MMP-2 and MMP-9, though it is unclear if this is linked to ATM. Here, we demonstrate ATM regulates aminopeptidase-N (CD13/APN/ANPEP) at the protein level. Positive correlation was seen between ATM activity and CD13 protein expression using both "wildtype" (WT) and knockout (KO) ataxia telangiectasia (AT) cells through western blotting; with the same effect shown when treating neuroblastoma cancer cell line SH-SY5Y, as well as AT-WT cells, with ATM inhibitor (ATMi; KU55933). However, qPCR along with publically available RNAseq data from Hu et al. (J. Clin. Invest., 2021, 131, e139333), demonstrated no change in mRNA levels of CD13, suggesting that ATM regulates CD13 levels via controlling protein degradation. This is further supported by the observation that incubation with proteasome inhibitors led to restoration of CD13 protein levels in cells treated with ATMi. Migration assays showed ATM and CD13 inhibition impairs migration, with no additional effect observed when combined. This suggests an epistatic effect, and that both proteins may be acting in the same signaling pathway that influences cell migration. This work indicates a novel functional interaction between ATM and CD13, suggesting ATM may negatively regulate the degradation of CD13, and subsequently cell migration.

Structure-based virtual screening of novel USP5 inhibitors targeting the zinc finger ubiquitin-binding domain

The equilibrium of cellular protein levels is pivotal for maintaining normal physiological functions. USP5 belongs to the deubiquitination enzyme (DUBs) family, controlling protein degradation and preserving cellular protein homeostasis. Aberrant expression of USP5 is implicated in a variety of diseases, including cancer, neurodegenerative diseases, and inflammatory diseases. In this paper, a multi-level virtual screening (VS) approach was employed to target the zinc finger ubiquitin-binding domain (ZnF-UBD) of USP5, leading to the identification of a highly promising candidate compound 0456–0049. Molecular dynamics (MD) simulations were then employed to assess the stability of complex binding and predict hotspot residues in interactions. The results indicated that the candidate stably binds to the ZnF-UBD of USP5 through crucial interactions with residues ARG221, TRP209, GLY220, ASN207, TYR261, TYR259, and MET266. Binding free energy calculations, along with umbrella sampling (US) simulations, underscored a superior binding affinity of the candidate relative to known inhibitors. Moreover, US simulations revealed conformational changes of USP5 during ligand dissociation. These insights provide a valuable foundation for the development of novel inhibitors targeting USP5.

Impaired systemic proteostasis and peripheral immune cell dysfunction in kidney diseases

ABSTRACT Kidney diseases, encompassing a spectrum of either acute or chronic disorders, manifest complex systemic repercussions beyond renal dysfunction. These include a reduced efficacy of tissue proteostasis mechanisms to control protein synthesis, folding, and degradation in both the renal and extra-renal compartments. Emerging research has revealed a pivotal interplay between proteostasis and immune regulation in the pathogenesis of kidney diseases, along with their cardiovascular, immunometabolic, and organ dysfunction symptoms. This review outlines the multifaceted connections between proteostasis dysregulation and immune dysfunction in kidney disease onset and progression. The crosstalk between the proteostasis network and immune cells orchestrates a bidirectional communication that amplifies pathological cascades, exacerbating kidney injury and impairing organ resilience. Furthermore, the article focuses on the implications of systemic proteostasis defects in modulating immune responses in extra-renal tissues underscore the systemic nature of kidney diseases. The disruption of proteostasis mechanisms triggers the release of damage-associated molecular patterns (DAMPs) and inflammatory mediators, perpetuating a state of heightened immune activation, contributing to systemic complications in affected individuals. Understanding the intricate interaction between proteostasis and immune regulation in kidney diseases, including both chronic and acute forms, promises novel therapeutic interventions. Targeting proteostasis pathways to restore cellular homeostasis and modulating immune responses could offer innovative strategies to mitigate renal damage and ameliorate systemic complications associated with kidney diseases. Harnessing this knowledge may also pave the way for the development of more efficient dialysis therapies and interventions with sorbents and hemoperfusion methods, to improve the clinical outcome of the patients.

From PROTAC to TPD: Advances and Opportunities in Targeted Protein Degradation.

PROTAC is a rapidly developing engineering technology for targeted protein degradation using the ubiquitin-proteasome system, which has promising applications for inflammatory diseases, neurodegenerative diseases, and malignant tumors. This paper gives a brief overview of the development and design principles of PROTAC, with a special focus on PROTAC-based explorations in recent years aimed at achieving controlled protein degradation and improving the bioavailability of PROTAC, as well as TPD technologies that use other pathways such as autophagy and lysosomes to achieve targeted protein degradation.

Radiation responsive PROTAC nanoparticles for tumor-specific proteolysis enhanced radiotherapy.

Proteolysis targeting chimeras (PROTACs) is a promising strategy for cancer therapy. However, the always-on bioactivity of PROTACs may lead to non-target toxicity, which restricts their antitumor performance. Here, we developed an X-ray radiation responsive PROTAC nanomicelle (RCNprotac) by covalently conjugating a reported small molecule PROTAC (MZ1) to hydrophilic PEG via a diselenide bond-containing carbon chain, which then self-assembled into a 141.80 ± 5.66 nm nanomicelle. The RCNprotac displayed no bioactivity during circulation due to the occupation of the hydroxyl group on the E3 ubiquitin ligand component and could effectively accumulate at the tumor site owing to the enhanced permeability and retention effect. Upon exposure to X-ray radiation, the radiation-sensitive diselenide bonds were broken to specifically release MZ1 for tumor BRD4 protein degradation. Furthermore, the reduction in the BRD4 protein level could increase the tumor's sensitivity to radiation. RCNprotac showed a synergistic enhancement of antitumor effects both in vitro and in vivo. We believe that this X-ray-responsive PROTAC nanomicelle could provide a new strategy for the X-ray-activated spatiotemporally controlled protein degradation and for the BRD4 proteolysis enhanced tumor radiosensitivity.

Near-infrared light controlled protein degradation by photo-caged lenalidomide and pomalidomide

Immunomodulatory drugs (e.g. thalidomide, lenalidomide and pomalidomide) have been proven highly successful in clinical treatment of multiple myeloma. However, systematic degradation of zinc finger transcriptional factors induced by these drugs could lead to severe systematic toxicity in patients. Previous reports of NVOC caged pomalidomide attempted to regulate its activity using UVA irradiation, but their application was limited by high cytotoxicity and low tissue penetration. Here, we reported red-shifted BODIPY caged lenalidomide and pomalidomide that enabled red-light controlled protein degradation with spatiotemporal precision.

Development of visible-light-activatable photocaged PROTACs

Photocaged proteolysis-targeting chimeras (PROTACs), which employ light as a stimulus to control protein degradation, have recently garnered considerable attention as both powerful chemical tools and a promising therapeutic strategy. However, the poor penetration depth of traditionally used ultraviolet light and the deficiency of alternative caging positions have restricted their applications in biological systems. By installing a diverse array of photocaged groups, with excitation wavelengths ranging from 365 nm to 405 nm, onto different positions of cereblon (CRBN) and Von Hippel-Lindau (VHL)-recruiting Brd4 degraders, we conducted the first comprehensive study on visible-light-activatable photocaged PROTACs to the best of our knowledge. We found the A2, A4 and B3 positions to be most effective at regulating the activity of the degraders, and to provide the resulting molecules (9–12 and 17) as potent visible-light-controlled degraders in live cells.

Urea Transporter B Is Produced in the Jk Null Blood Group Yet Fails to Integrate into the Red Cell Membrane Impacting Erythropoiesis and Erythrocytes

The Kidd (Jk) blood group is carried by an integral membrane glycoprotein, urea transporter B (UT-B), which transports urea through the red blood cell (RBC) membrane. The absence of both Jk a and Jk b antigens, designated phenotypically as Jk(a-b-) (Jk null), is very rare with a significantly increased prevalence in certain ethnicities, most abundantly within the Polynesian population where it is found at 0.9% prevalence. Provision of blood for patients requiring this phenotype is challenging. In Australia, there are only 15 active Jk null blood donors. Individuals of the Jk null phenotype have been shown to have suboptimal urine concentrating ability but erythrocytes appear phenotypically normal with shape, size and lifespan within normal reference indices (Lawicki et al., Transfus Med Rev, 2017). Where UT-B integrates in the RBC membrane is unknown although protein 4.1R deficiency is reported to lead to the absence of UT-B (Azouzi et al., Br J Haematol, 2015). Our aim was to identify and characterise the molecular basis for the Jk null phenotype in Australian blood donors and decipher the biological impact of the Jk null phenotype using our ex vivo model of erythropoiesis to help inform clinical transfusion practice. Massively parallel sequencing showed that all blood donors (n=9) were homozygous for JK c.342-1G>A (splice site variant) and c.838G>A polymorphisms resulting in the JK*02N.01/*02N.01 genotype. This genotype predicts a Jk null phenotype, and a truncated UT-B protein caused by the formation of a premature stop codon. The assumption from the genomics data is that the putative truncated UT-B protein would not be translated. To test this hypothesis we examined the RBCs of Jk null individuals and confirmed the lack of UT-B expression on the RBC surface by live flow cytometry and confocal imaging. In control RBCs UT-B is seen in discrete microdomains on the RBC surface by live confocal microscopy. To establish the expression of UT-B during erythropoiesis we cultured CD34+ cells from control and Jk null buffy coats after whole blood donation. Flow cytometry and confocal microscopy of control cells identified that UT-B is expressed intracellularly and at the cell surface in the developing erythroid cells. Notably we identified that the UT-B protein can also be found intracellularly in developing Jk null erythroid cells but is not detectable in the plasma membrane. In Jk null reticulocytes, UT-B colocalises with markers of protein degradation and destruction implying that the truncated UT-B protein is quickly removed by controlled protein degradation pathways. Quantitative proteomics was used to assess the global effect of the Jk null phenotype on the mature RBC membrane proteome. These data confirmed the absence of UT-B but also uncovered that in mature Jk null RBCs, RBC proteins involved in solute transport (aquaporin-1, stomatin) and membrane-cytoskeleton stabilisation (alpha and beta adducins, and dematin) are significantly downregulated. In contrast, actin binding proteins including tropomyosin and myosin IIA were upregulated in Jk null RBCs. The proteomics data also revealed that numerous major plasma proteins including alpha-2-macroglobulin, hemopexin and immunoglobulins are associated exclusively with Jk null RBCs. These findings imply that the lack of functional, membrane embedded UT-B induces various compensatory mechanisms in the Jk null RBCs membrane. In conclusion, we show that a truncated UT-B protein is produced in Jk null erythroid cells but fails to mature into a RBC membrane integrated, functional glycoprotein. Our data also suggest that the absence of a functional UT-B glycoprotein on the RBC surface affects the RBC membrane and cytoskeletal proteome and its interaction with major plasma glycoproteins.

Apical PAR protein caps orient the mitotic spindle in C. elegans early embryos

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases, it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C.elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact-free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, isolated single blastomeres lacking cell contacts are able to break symmetry and form PAR-3/atypical protein kinase C (aPKC) caps. Polarity caps form independently of actomyosin flows and microtubules and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

Stimuli‐Responsive PROTACs for Controlled Protein Degradation

AbstractProteolysis Targeting Chimeras (PROTACs) represent a promising therapeutic modality to address undruggable and resistant issues in drug discovery. However, potential on‐target toxicity remains clinically challenging. We developed a generalized caging strategy to synthesize a series of stimuli‐responsive PROTACs (sr‐PROTACs) with diverse molecular blocks bearing robust and cleavable linkers, presenting “turn on” features in manipulating protein degradation. By leveraging pathological cues, such as elevated ROS, phosphatase, H2S, or hypoxia, and external triggers, such as ultraviolet light, X‐Ray, or bioorthogonal reagents, we achieved site‐specific activation and traceless release of original PROTACs through de‐caging and subsequent self‐immolative cleavage, realizing selective uptake and controlled protein degradation in vitro. An in vivo study revealed that two sr‐PROTACs with phosphate‐ and fluorine‐containing cages exhibited high solubility and long plasma exposure, which were specifically activated by tumor overexpressing phosphatase or low dosage of X‐Ray irradiation in situ, leading to efficient protein degradation and potent tumor remission. With more reactive biomarkers to be screened from clinical practice, our caging library could provide a general tool to design activatable PROTACs, prodrugs, antibody‐drug conjugates, and smart biomaterials for personalized treatment, tissue engineering or regenerative medicine.