BET inhibition induces synthetic lethality in PTEN deficient colorectal cancers via dual action on p21CIP1/WAF1.

Loss of PTEN tumor suppressor is an important event during colorectal cancer (CRC) development and is a target for therapeutic exploitation. This study reports that bromodomain and extra-terminal motif (BET) is a synthetic lethal partner of PTEN in CRC. BET inhibition (BETi) selectively induced G1 cell cycle arrest and apoptosis in PTEN-/- CRC. Further, BETi selectively and dose-dependently suppressed the growth of PTEN-/- CRC tumor xenografts in mice and patient-derived organoids. Mechanistically, PTEN-deficient CRC cells elevated the level of cytoplasmic p21CIP1/WAF1 that is hyper-phosphorylated at Thr145 by AKT. BETi suppressed AKT activation in PTEN-deficient CRC cells, followed by the reduction in p21 phosphorylation at Thr145, thereby promoting its nuclear translocation. In addition, BETi suppressed MYC level and this in turn increased the total p21 level in the nuclei. Over-expression of a phospho-mimetic p21 mutant (T145D) significantly rescued the BETi effect on PTEN-deficient CRC. These results suggest that BETi has a dual action on p21: elevating the level of p21 by inhibiting MYC and converting the oncogenic (cytoplasmic) p21 into the tumor-suppressive (nuclear) p21 by inhibiting AKT. Taken together, this study identified the synthetic lethal interaction between PTEN and BET, and provides a potential actionable target for CRC with PTEN loss.

Analysis of the interaction between the ORF42 and ORF55 proteins encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The tegument is a structure that is unique to herpesviruses that includes host and viral proteins, including the viral ORF42 and ORF55 proteins. Alphaherpesvirus tegument proteins have been well studied, but much is unknown regarding KSHV. Here, we report an interaction between the ORF42 and ORF55 proteins. ORF55 interacted with and recruited ORF42 from the nucleus to the cytoplasm. When ORF42 and ORF55 were expressed simultaneously in cultured cells, the expression level of these two viral proteins was higher than when either was expressed independently. ORF55, but not ORF42, was polyubiquitinated, suggesting that an unidentified regulatory mechanism may be present. A recombinant virus with an ectopic stop codon in ORF42 exhibited normal replication of genomic DNA, but fewer virus particles were released with the recombinant than with the wild-type virus. A unique R136Q mutation in ORF42, which is found in a KSHV strain that is prevalent on Miyako Island, Okinawa Prefecture, Japan, further increased the expression of ORF42 and ORF55 when these proteins were expressed simultaneously. However, the ORF42 R136Q mutation did not affect the localization pattern of ORF42 itself or of ORF55. In addition, experiments with a recombinant virus possessing the ORF42 R136Q mutation showed lower levels of production of the mutant virus than of the wild-type virus, despite similar levels of genome replication. We suggest that the R136Q mutation in ORF42 plays an important role in ORF55 protein expression and virus production.

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa.

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Association between Parkinson's Disease and Cancer: New Findings and Possible Mediators.

Epidemiological evidence points to an inverse association between Parkinson's disease (PD) and almost all cancers except melanoma, for which this association is positive. The results of multiple studies have demonstrated that patients with PD are at reduced risk for the majority of neoplasms. Several potential biological explanations exist for the inverse relationship between cancer and PD. Recent results identified several PD-associated proteins and factors mediating cancer development and cancer-associated factors affecting PD. Accumulating data point to the role of genetic traits, members of the synuclein family, neurotrophic factors, the ubiquitin-proteasome system, circulating melatonin, and transcription factors as mediators. Here, we present recent data about shared pathogenetic factors and mediators that might be involved in the association between these two diseases. We discuss how these factors, individually or in combination, may be involved in pathology, serve as links between PD and cancer, and affect the prevalence of these disorders. Identification of these factors and investigation of their mechanisms of action would lead to the discovery of new targets for the treatment of both diseases.

Domains in Action: Understanding Ddi1's Diverse Functions in the Ubiquitin-Proteasome System.

The ubiquitin-proteasome system (UPS) is a pivotal cellular mechanism responsible for the selective degradation of proteins, playing an essential role in proteostasis, protein quality control, and regulating various cellular processes, with ubiquitin marking proteins for degradation through a complex, multi-stage process. The shuttle proteins family is a very unique group of proteins that plays an important role in the ubiquitin-proteasome system. Ddi1, Dsk2, and Rad23 are shuttle factors that bind ubiquitinated substrates and deliver them to the 26S proteasome. Besides mediating the delivery of ubiquitinated proteins, they are also involved in many other biological processes. Ddi1, the least-studied shuttle protein, exhibits unique physicochemical properties that allow it to play non-canonical functions in the cells. It regulates cell cycle progression and response to proteasome inhibition and defines MAT type of yeast cells. The Ddi1 contains UBL and UBA domains, which are crucial for binding to proteasome receptors and ubiquitin respectively, but also an additional domain called RVP. Additionally, much evidence has been provided to question whether Ddi1 is a classical shuttle protein. For many years, the true nature of this protein remained unclear. Here, we highlight the recent discoveries, which shed new light on the structure and biological functions of the Ddi1 protein.

EH domain-containing protein 2 (EHD2): Overview, biological function, and therapeutic potential.

EH domain-containing protein 2 (EHD2) is a member of the EHD protein family and is mainly located in the plasma membrane, but can also be found in the cytoplasm and endosomes. EHD2 is also a nuclear-cytoplasmic shuttle protein. After entering the cell nuclear, EHD2 acts as a corepressor of transcription to inhibit gene transcription. EHD2 regulates a series of biological processes. As a key regulator of endocytic transport, EHD2 is involved in the formation and maintenance of endosomal tubules and vesicles, which are critical for the intracellular transport of proteins and other substances. The N-terminal of EHD2 is attached to the cell membrane, while its C-terminal binds to the actin-binding protein. After binding, EHD2 connects with the actin cytoskeleton, forming the curvature of the membrane and promoting cell endocytosis. EHD2 is also associated with membrane protein trafficking and receptor signaling, as well as in glucose metabolism and lipid metabolism. In this review, we highlight the recent advances in the function of EHD2 in various cellular processes and its potential implications in human diseases such as cancer and metabolic disease. We also discussed the prospects for the future of EHD2. EHD2 has a broad prospect as a therapeutic target for a variety of diseases. Further research is needed to explore its mechanism, which could pave the way for the development of targeted treatments.

Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders.

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focuses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease.

Differential Interactions of the Proteasome Inhibitor PI31 with Constitutive and Immuno-20S Proteasomes.

PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes have revealed that the natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the 20S proteasome and interacts directly with the proteasome's multiple catalytic threonine residues in a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of the 20S proteasome (termed "immuno-proteasome") that features genetically and functionally distinct catalytic subunits. The effect of PI31 on immuno-proteasome function is unknown. We examine the relative inhibitory effects of PI31 on purified constitutive (20Sc) and immuno-(20Si) 20S proteasomes in vitro and show that PI31 inhibits 20Si hydrolytic activity to a significantly lesser degree than that of 20Sc. Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31 and this effect contributes to the reduced inhibitory activity of PI31 toward 20Si. Conversely, loss of 20Sc inhibition by PI31 point mutants leads to PI31 degradation by 20Sc. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.

Animal Models of FUS-Proteinopathy: A Systematic Review.

Mutations that disrupt the function of the DNA/RNA-binding protein FUS could cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. One of the key features in ALS pathogenesis is the formation of insoluble protein aggregates containing aberrant isoforms of the FUS protein in the cytoplasm of upper and lower motor neurons. Reproduction of human pathology in animal models is the main tool for studying FUS-associated pathology and searching for potential therapeutic agents for ALS treatment. In this review, we provide a systematic analysis of the role of FUS protein in ALS pathogenesis and an overview of the results of modelling FUS-proteinopathy in animals.

Biomolecular Condensates: Structure, Functions, Methods of Research.

The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid-liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless microcompartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogenesis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular condensates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo.

Navigating the signaling landscape of Ralstonia solanacearum: a study of bacterial two-component systems.

Ralstonia solanacearum, the bacterium that causes bacterial wilt, is a destructive phytopathogen that can infect over 450 different plant species. Several agriculturally significant crop plants, including eggplant, tomato, pepper, potato, and ginger, are highly susceptible to this plant disease, which has a global impact on crop quality and yield. There is currently no known preventive method that works well for bacterial wilt. Bacteria use two-component systems (TCSs) to sense their environment constantly and react appropriately. This is achieved by an extracellular sensor kinase (SK) capable of sensing a suitable signal and a cytoplasmic response regulator (RR) which gives a downstream response. Moreover, our investigation revealed that R. solanacearum GMI1000 possesses a substantial count of TCSs, specifically comprising 36 RRs and 27 SKs. While TCSs are known targets for various human pathogenic bacteria, such as Salmonella, the role of TCSs in R. solanacearum remains largely unexplored in this context. Notably, numerous inhibitors targeting TCSs have been identified, including GHL (Gyrase, Hsp, and MutL) compounds, Walk inhibitors, and anti-TCS medications like Radicicol. Consequently, the investigation into the involvement of TCSs in virulence and pathogenesis has gained traction; however, further research is imperative to ascertain whether TCSs could potentially supplant conventional anti-wilt therapies. This review delves into the prospective utilization of TCSs as an alternative anti-wilt therapy, focusing on the lethal phytopathogen R. solanacearum.

LLPS of FXR proteins drives replication organelle clustering for ß-coronaviral proliferation.

ß-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid-liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

Smaug regulates germ plasm assembly and primordial germ cell number in Drosophila embryos.

During Drosophila oogenesis, the Oskar (OSK) RNA binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here, we identify mechanisms that subsequently regulate germ plasm assembly in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk messenger RNA (mRNA) as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNA results in excess translation of these transcripts in the germ plasm, accumulation of excess germ plasm, and budding of excess primordial germ cells (PGCs). Therefore, SMG triggers a posttranscriptional regulatory pathway that attenuates the amount of germ plasm in embryos to modulate the number of PGCs.

Immune patterns of cuproptosis in ischemic heart failure: A transcriptome analysis.

Cuproptosis is a recently discovered programmed cell death pattern that affects the tricarboxylic acid (TCA) cycle by disrupting the lipoylation of pyruvate dehydrogenase (PDH) complex components. However, the role of cuproptosis in the progression of ischemic heart failure (IHF) has not been investigated. In this study, we investigated the expression of 10 cuproptosis-related genes in samples from both healthy individuals and those with IHF. Utilizing these differential gene expressions, we developed a risk prediction model that effectively distinguished healthy and IHF samples. Furthermore, we conducted a comprehensive evaluation of the association between cuproptosis and the immune microenvironment in IHF, encompassing infiltrated immunocytes, immune reaction gene-sets and human leukocyte antigen (HLA) genes. Moreover, we identified two different cuproptosis-mediated expression patterns in IHF and explored the immune characteristics associated with each pattern. In conclusion, this study elucidates the significant influence of cuproptosis on the immune microenvironment in ischemic heart failure (IHF), providing valuable insights for future mechanistic research exploring the association between cuproptosis and IHF.

Basement membrane diversification relies on two competitive secretory routes defined by Rab10 and Rab8 and modulated by dystrophin and the exocyst complex.

The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. Drosophila melanogaster follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.

An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans.

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in Caenorhabditis elegans has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic cells. We have tagged endogenous transcripts with MS2 hairpins in the 3' untranslated region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs for dlg-1 fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, and dlg-1 RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.

Characteristic and Import Mechanism of Protein Nuclear Translocation.

Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and nucleoplasm is crucial for many physiological processes. Macromolecular protein transport into the nucleus requires assistance from the nuclear transport system. These proteins typically contain a nuclear localisation sequence that guides them to enter the nucleus. Understanding the mechanism of nuclear import of macromolecular proteins is important for comprehending cellular processes. Investigation of disease-related alterations can facilitate the development of novel therapeutic strategies and provide additional evidence for clinical trials. This review provides an overview of the proteins involved in nuclear transport and the mechanisms underlying macromolecular protein transport.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

RNA damage compartmentalization by DHX9 stress granules.

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development.

Embryonic genome activation (EGA) marks the transition from dependence on maternal transcripts to an embryonic transcriptional program. The precise temporal regulation of gene expression, specifically the silencing of the Dux/murine endogenous retrovirus type L (MERVL) program during late 2-cell interphase, is crucial for developmental progression in mouse embryos. How this finely tuned regulation is achieved within this specific window is poorly understood. Here, using particle-tracking microrheology throughout the mouse oocyte-to-embryo transition, we identify a surge in cytoplasmic viscosity specific to late 2-cell interphase brought about by high microtubule and endomembrane density. Importantly, preventing the rise in 2-cell viscosity severely impairs nuclear reorganization, resulting in a persistently open chromatin configuration and failure to silence Dux/MERVL. This, in turn, derails embryo development beyond the 2- and 4-cell stages. Our findings reveal a mechanical role of the cytoplasm in regulating Dux/MERVL repression via nuclear remodeling during a temporally confined period in late 2-cell interphase.