Caspase-2 protects against ferroptotic cell death.

Caspase-2, one of the most evolutionarily conserved members of the caspase family, is an important regulator of the cellular response to oxidative stress. Given that ferroptosis is suppressed by antioxidant defense pathways, such as that involving selenoenzyme glutathione peroxidase 4 (GPX4), we hypothesized that caspase-2 may play a role in regulating ferroptosis. This study provides the first demonstration of an important and unprecedented function of caspase-2 in protecting cancer cells from undergoing ferroptotic cell death. Specifically, we show that depletion of caspase-2 leads to the downregulation of stress response genes including SESN2, HMOX1, SLC7A11, and sensitizes mutant-p53 cancer cells to cell death induced by various ferroptosis-inducing compounds. Importantly, the canonical catalytic activity of caspase-2 is not required for its role and suggests that caspase-2 regulates ferroptosis via non-proteolytic interaction with other proteins. Using an unbiased BioID proteomics screen, we identified novel caspase-2 interacting proteins (including heat shock proteins and co-chaperones) that regulate cellular responses to stress. Finally, we demonstrate that caspase-2 limits chaperone-mediated autophagic degradation of GPX4 to promote the survival of mutant-p53 cancer cells. In conclusion, we document a novel role for caspase-2 as a negative regulator of ferroptosis in cells with mutant p53. Our results provide evidence for a novel function of caspase-2 in cell death regulation and open potential new avenues to exploit ferroptosis in cancer therapy.

Understanding Developmental Cell Death Using Drosophila as a Model System.

Cell death plays an essential function in organismal development, wellbeing, and ageing. Many types of cell deaths have been described in the past 30 years. Among these, apoptosis remains the most conserved type of cell death in metazoans and the most common mechanism for deleting unwanted cells. Other types of cell deaths that often play roles in specific contexts or upon pathological insults can be classed under variant forms of cell death and programmed necrosis. Studies in Drosophila have contributed significantly to the understanding and regulation of apoptosis pathways. In addition to this, Drosophila has also served as an essential model to study the genetic basis of autophagy-dependent cell death (ADCD) and other relatively rare types of context-dependent cell deaths. Here, we summarise what is known about apoptosis, ADCD, and other context-specific variant cell death pathways in Drosophila, with a focus on developmental cell death.

The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.

Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic vesicle size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified detour interacting proteins including HOPS subunits, deep orange (dor/VPS18), Vacuolar protein sorting 16A (VPS16A), and light (lt/VPS41) and found that detour promotes their ubiquitination. The detour mutant accumulated autophagy-related proteins in young adults, displayed premature ageing, impaired motor function, and activation of innate immunity. Collectively, our findings suggest a role for detour in autophagy, likely through regulation of HOPS complex, with implications for healthy aging.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling.

Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer+ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis.Abbreviations: AMPK, 5'-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A1; CQ, chloroquine; DMEM, Dulbecco's minimum essential medium; DPBS, Dulbecco's phosphate-buffered saline; EBSS, Earle's balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

An insulin-regulated arrestin domain protein controls hepatic glucagon action.

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.

Receptor Recycling by Retromer.

The highly conserved retromer complex controls the fate of hundreds of receptors that pass through the endolysosomal system and is a central regulatory node for diverse metabolic programs. More than 20 years ago, retromer was discovered as an essential regulator of endosome-to-Golgi transport in yeast; since then, significant progress has been made to characterize how metazoan retromer components assemble to enable its engagement with endosomal membranes, where it sorts cargo receptors from endosomes to the trans-Golgi network or plasma membrane through recognition of sorting motifs in their cytoplasmic tails. In this review, we examine retromer regulation by exploring its assembled structure with an emphasis on how a range of adaptor proteins shape the process of receptor trafficking. Specifically, we focus on how retromer is recruited to endosomes, selects cargoes, and generates tubulovesicular carriers that deliver cargoes to target membranes. We also examine how cells adapt to distinct metabolic states by coordinating retromer expression and function. We contrast similarities and differences between retromer and its related complexes: retriever and commander/CCC, as well as their interplay in receptor trafficking. We elucidate how loss of retromer regulation is central to the pathology of various neurogenerative and metabolic diseases, as well as microbial infections, and highlight both opportunities and cautions for therapeutics that target retromer. Finally, with a focus on understanding the mechanisms that govern retromer regulation, we outline new directions for the field moving forward.

TP53 mutation variant allele frequency of ≥10% is associated with poor prognosis in therapy-related myeloid neoplasms.

Revised diagnostic criteria for myeloid neoplasms (MN) issued by the International Consensus Classification (ICC) and the World Health Organization (WHO) recommended major change pertaining to TP53-mutated (TP53mut) MN. However, these assertions have not been specifically examined in therapy-related myeloid neoplasm (t-MN), a subset enriched with TP53mut. We analyzed 488 t-MN patients for TP53mut. At least one TP53mut with variant allele frequency (VAF) ≥ 2% with or without loss of TP53 locus was noted in 182 (37.3%) patients and 88.2% of TP53mut t-MN had a VAF ≥10%. TP53mut t-MN with VAF ≥ 10% had a distinct clinical and biological profile compared to both TP53mut VAF < 10% and wild-type TP53 (TP53wt) cases. Notably, TP53mut VAF ≥ 10% had a significantly shorter survival compared to TP53wt (8.3 vs. 21.6 months; P < 0.001), while the survival of TP53mut VAF < 10% was comparable to TP53wt. Within TP53mut VAF ≥ 10% cohort, the inferior outcomes persisted irrespective of the single- or multi-hit status, co-mutation pattern, or treatments received. Finally, survival of TP53mut patients was poor across all the blast categories and MDS patients with >10% blasts had inferior survival compared to <5%. In summary, TP53mut VAF ≥10% signified a clinically and molecularly homogenous cohort regardless of the allelic status.

Ganglion Impar Block: A Magic Bullet to Fix Idiopathic Coccygodynia.

Coccygodynia (coccydynia) is a painful condition of the perineum in the region of the tailbone or coccyx, aggravated by sitting on hard surfaces. It is frequently associated with injuries to the coccyx following direct trauma. Nevertheless, idiopathic coccygodynia without antecedent trauma history is not uncommon. Most of these patients respond to anti-inflammatory medications and physical therapy. Those who are unresponsive may require additional intervention for pain relief. Blockade of ganglion impar, the terminal end of the pelvic sympathetic chain, can dramatically alleviate the pain in patients suffering from coccygodynia. In the current case series, four patients in the age range of 21 to 69 years suffering from chronic idiopathic coccygodynia (female: male ratio of 1:1) were treated with ganglion impar block. All four patients received a course of medical management, and two of the patients additionally received local infiltration of the coccyx before ganglion impar block administration. The block was performed with fluoroscopy guidance by either the trans-sacrococcygeal joint approach or the intra-coccygeal joint approach. The pre-intervention average numeric rating pain score (NRS) was 7.5. After a single ganglion impar block intervention, all four patients experienced complete pain relief (NRS=0). No patients required a repeat injection, and all were pain-free for the entire one-year follow-up period.

A Rare Case of Kostmann Syndrome Presenting Difficult Airway Challenges and Patient Preparedness for Anesthesiologists.

Severe congenital neutropenia (SCN), commonly known as the Kostmann syndrome, is a rare and complex set of disorders defined by a lack of neutrophil maturation in the bone marrow, leading to life-threatening complications. This case report discusses a young adult patient scheduled for elective laparoscopic cholecystectomy. The patient presented with skin lesions which are a common scenario of Kostmann syndrome, but along with that, our patient posed challenges of short neck, limited neck extension, and gynecomastia. These additional conditions dramatically increased the challenges for anesthesiologists to address the anticipated difficult airway. The anticipated difficult airway challenges were handled by following the protocols of difficult airway guidelines 2022.

A senescence stress secretome is a hallmark of therapy-related myeloid neoplasm stromal tissue occurring soon after cytotoxic exposure.

Therapy-related myeloid neoplasm (tMN) is considered a direct consequence of DNA damage in hematopoietic stem cells. Despite increasing recognition that altered stroma can also drive leukemogenesis, the functional biology of the tMN microenvironment remains unknown. We performed multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterization of bone marrow stromal cells from tMN patients. Critically, we also compared (i) patients with myeloid neoplasm and another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm, and (iii) age-matched controls to decipher the microenvironmental changes induced by cytotoxics vs. neoplasia. Strikingly, tMN exhibited a profoundly senescent phenotype with induction of CDKN1A and ß-Galactosidase, defective phenotype, and proliferation. Moreover, tMN stroma showed delayed DNA repair and defective adipogenesis. Despite their dormant state, tMN stromal cells were metabolically highly active with a switch toward glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. Our data underscores a role of senescence in the pathogenesis of tMN and provide a valuable resource for future therapeutics.

Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells.

The ubiquitin ligase NEDD4 promotes neural crest cell (NCC) survival and stem-cell like properties to regulate craniofacial and peripheral nervous system development. However, how ubiquitination and NEDD4 control NCC development remains unknown. Here we combine quantitative analysis of the proteome, transcriptome and ubiquitinome to identify key developmental signalling pathways that are regulated by NEDD4. We report 276 NEDD4 targets in NCCs and show that loss of NEDD4 leads to a pronounced global reduction in specific ubiquitin lysine linkages. We further show that NEDD4 contributes to the regulation of the NCC actin cytoskeleton by controlling ubiquitination and turnover of Profilin 1 to modulate filamentous actin polymerization. Taken together, our data provide insights into how NEDD4-mediated ubiquitination coordinates key regulatory processes during NCC development.

Anesthetic Considerations for Elective Laparoscopic Cholecystectomy in a Patient With Previous Pneumonectomy.

Patients presenting for surgery after pneumonectomy pose significant challenges to anesthesiologists. The disease process necessitating pneumonectomy may involve the surviving lung too. Cholecystectomy is a major surgery, and the open approach has significant risks of post-operative pulmonary complications in these patients partly owing to the large incision and postoperative atelectasis, associated with inadequate post-operative analgesia. Contemplating a laparoscopic procedure in patients with a single, possibly damaged lung, involves a good understanding of the physiology of the single lung as well as the challenges posed by capnoperitoneum. Here, we present a case of a female with a history of previous pneumonectomy undergoing laparoscopic cholecystectomy. There are very few reports of patients after pneumonectomy who have subsequently undergone a laparoscopic cholecystectomy successfully and this report highlights some crucial factors to be kept in mind during anesthetic management of such patients.

K-29 linked ubiquitination of Arrdc4 regulates its function in extracellular vesicle biogenesis.

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT , and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R . K270 was found to be ubiquitinated with K-29 polyubiquitin chains by the ubiquitin ligase Nedd4-2. Thus, our results uncover a novel role of K-29 polyubiquitin chains in Arrdc4-mediated EV biogenesis and protein trafficking.

Loss of NEDD4 causes complete XY gonadal sex reversal in mice.

Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, maleness is driven by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and ß-catenin. Here, we report a hitherto uncharacterised role for the ubiquitin-protein ligase NEDD4 in this process. XY Nedd4-deficient mice exhibit complete male-to-female gonadal sex reversal shown by the ectopic upregulation of Foxl2 expression at the time of gonadal sex determination as well as insufficient upregulation of Sox9. This sex reversal extends to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4-deficient mice when compared to both XY and XX controls. Further, Nedd4-/- mice exhibit reduced gonadal precursor cell formation and gonadal size as a result of reduced proliferation within the developing gonad as well as reduced Nr5a1 expression. Together, these results establish an essential role for NEDD4 in XY gonadal sex determination and development and suggest a potential role for NEDD4 in orchestrating these cell fate decisions through the suppression of the female pathway to ensure proper testis differentiation.

The role of caspases as executioners of apoptosis.

Caspases are a family of cysteine aspartyl proteases mostly involved in the execution of apoptotic cell death and in regulating inflammation. This article focuses primarily on the evolutionarily conserved function of caspases in apoptosis. We summarise which caspases are involved in apoptosis, how they are activated and regulated, and what substrates they target for cleavage to orchestrate programmed cell death by apoptosis.

Moderate to Severe Thrombocytopenia in Four Pregnant Women With Asymptomatic COVID-19 Infection.

Coronavirus disease 2019 (COVID-19) pandemic is one of the biggest healthcare crises faced globally. Since its emergence, uncertainty about its progress and treatment options has challenged clinicians around the world. Pregnant women infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection pose a higher challenge due to the concerns of an already altered immune system during pregnancy and the disease's effect on the fetus. Thrombocytopenia is associated frequently with moderate to severe coronavirus disease and is also an established marker of worsening of the disease. However, it is infrequently seen in mild or asymptomatic cases. Neuraxial anesthesia is the preferred choice of anesthesia in COVID-19 positive patients but thrombocytopenia in a parturient with coronavirus disease can cause a dilemma for the obstetric anesthesiologist. Here we describe the management of four pregnant women with asymptomatic COVID-19 disease who had moderate to severe thrombocytopenia. These cases highlight the importance of careful monitoring of the platelet count of pregnant women with COVID-19 infection even if asymptomatic.

ATG8ylation of proteins: A way to cope with cell stress?

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed "ATG8ylation," which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.