Advancements of prodrug technologies for enhanced drug selectivity in pharmacotherapies.

The therapeutic effects of many pharmacotherapies have been explored, but disadvantages such as low drug specificity, drug resistance and side effects makes their effective delivery to target sites a great challenge. Consequently, a distinctive prodrug-based technology have emerged as an effective treatments because of their distinctive advantages, such as high drug loading capacity, precise targeting, reduced side effects and spatial and temporal controllability. In particular, the use of gamma/X-ray-mediated strategies in radiotherapy is a new strategy that could enable the precise drug release from implanted devices. This review presents readers with the current state of prodrug therapy and reports the design protocols of rational and effective prodrugs for clinical use.

Effect of Terminal Phosphate Groups on Collisional Dissociation of RNA Oligonucleotide Anions.

The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.

Germ granule compartments coordinate specialized small RNA production.

Germ granules are biomolecular condensates present in most animal germ cells. One function of germ granules is to help maintain germ cell totipotency by organizing mRNA regulatory machinery, including small RNA-based gene regulatory pathways. The C. elegans germ granule is compartmentalized into multiple subcompartments whose biological functions are largely unknown. Here, we identify an uncharted subcompartment of the C. elegans germ granule, which we term the E granule. The E granule is nonrandomly positioned within the germ granule. We identify five proteins that localize to the E granule, including the RNA-dependent RNA polymerase (RdRP) EGO-1, the Dicer-related helicase DRH-3, the Tudor domain-containing protein EKL-1, and two intrinsically disordered proteins, EGC-1 and ELLI-1. Localization of EGO-1 to the E granule enables synthesis of a specialized class of 22G RNAs, which derive exclusively from 5' regions of a subset of germline-expressed mRNAs. Defects in E granule assembly elicit disordered production of endogenous siRNAs, which disturbs fertility and the RNAi response. Our results define a distinct subcompartment of the C. elegans germ granule and suggest that one function of germ granule compartmentalization is to facilitate the localized production of specialized classes of small regulatory RNAs.

Development of a Quorum Sensing-Mediated Bacterial Autolytic System in Escherichia coli for Automatic Release of Intracellular Products.

Escherichia coli, one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. De novo engineering of gene regulation circuits and cell density-controlled E. coli cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an E. coli autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from Pseudoalteromonas into E. coli cells. The results showed that the E. coli QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of E. coli cell density, which offers an environmentally-friendly, economical, efficient, and flexible E. coli lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in E. coli for medical and industrial applications.

ppGpp is a dual-role regulator involved in balancing iron absorption and prodiginine biosynthesis in Pseudoalteromonas.

Iron is an essential element for microbial survival and secondary metabolism. However, excess iron availability and overloaded secondary metabolites can hinder microbial growth and survival. Microorganisms must tightly control iron homeostasis and secondary metabolism. Our previous studies have found that the stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis by activating iron uptake in Pseudoalteromonas sp. strain R3. It is believed that the interaction between SspA and the small nucleotide ppGpp is important for iron to exert regulation functions. However, the roles of ppGpp in iron absorption and prodiginine biosynthesis, and the underlying relationship between ppGpp and SspA in strain R3 remain unclear. In this study, we found that ppGpp accumulation in strain R3 could be induced by limiting iron. In addition, ppGpp not only positively regulated iron uptake and prodiginine biosynthesis via increasing the SspA level but also directly repressed iron uptake and prodiginine biosynthesis independent of SspA, highlighting the finding that ppGpp can stabilize both iron levels and prodiginine production. Notably, the abolishment of ppGpp significantly increased prodiginine production, thus providing a theoretical basis for manipulating prodiginine production in the future. This dynamic ppGpp-mediated interaction between iron uptake and prodiginine biosynthesis has significant implications for understanding the roles of nutrient uptake and secondary metabolism for the survival of bacteria in unfavorable environments.

Diagnostic Value of Clear Cell Likelihood Score v1.0 and v2.0 for Common Subtypes of Small Renal Masses: A Multicenter Comparative Study.

BACKGROUND: Clear cell likelihood score (ccLS) is reliable for diagnosing small renal masses (SRMs). However, the diagnostic value of Clear cell likelihood score version 1.0 (ccLS v1.0) and v2.0 for common subtypes of SRMs might be a potential score extension. PURPOSE: To compare the diagnostic performance and interobserver agreement of ccLS v1.0 and v2.0 for characterizing five common subtypes of SRMs. STUDY TYPE: Retrospective. POPULATION: 797 patients (563 males, 234 females; mean age, 53 ± 12 years) with 867 histologically proven renal masses. FIELD STRENGTH/SEQUENCES: 3.0 and 1.5 T/T2 weighted imaging, T1 weighted imaging, diffusion-weighted imaging, a dual-echo chemical shift (in- and opposed-phase) T1 weighted imaging, multiphase dynamic contrast-enhanced imaging. ASSESSMENT: Six abdominal radiologists were trained in the ccLS algorithm and independently scored each SRM using ccLS v1.0 and v2.0, respectively. All SRMs had definite pathological results. The pooled area under curve (AUC), accuracy, sensitivity, and specificity were calculated to evaluate the diagnostic performance of ccLS v1.0 and v2.0 for characterizing common subtypes of SRMs. The average κ values were calculated to evaluate the interobserver agreement of the two scoring versions. STATISTICAL TESTS: Random-effects logistic regression; Receiver operating characteristic analysis; DeLong test; Weighted Kappa test; Z test. The statistical significance level was P < 0.05. RESULTS: The pooled AUCs of clear cell likelihood score version 2.0 (ccLS v2.0) were statistically superior to those of ccLS v1.0 for diagnosing clear cell renal cell carcinoma (ccRCC) (0.907 vs. 0.851), papillary renal cell carcinoma (pRCC) (0.926 vs. 0.888), renal oncocytoma (RO) (0.745 vs. 0.679), and angiomyolipoma without visible fat (AMLwvf) (0.826 vs. 0.766). Interobserver agreement for SRMs between ccLS v1.0 and v2.0 is comparable and was not statistically significant (P = 0.993). CONCLUSION: The diagnostic performance of ccLS v2.0 surpasses that of ccLS v1.0 for characterizing ccRCC, pRCC, RO, and AMLwvf. Especially, the standardized algorithm has optimal performance for ccRCC and pRCC. ccLS has potential as a supportive clinical tool. EVIDENCE LEVEL: 4. TECHNICAL EFFICACY: Stage 2.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Regulation of fadR on the ROS defense mechanism in Shewanalla oneidensis.

Protein FadR is known as a fatty acid metabolism global regulator that sustains cell envelope integrity by changing the profile of fatty acid. Here, we present its unique participation in the defense against reactive oxygen species (ROS) in the bacterium. FadR contributes to defending extracellular ROS by maintaining the permeability of the cell membrane. It also facilitates the ROS detoxification process by increasing the expression of ROS neutralizers (KatB, KatG, and AhpCF). FadR also represses the leakage of ROS by alleviating the respiratory action conducted by terminal cytochrome cbb3-type heme-copper oxidases (ccoNOQP). These findings suggest that FadR plays a comprehensive role in modulating the bacterial oxidative stress response, instead of merely strengthening the cellular barrier against the environment. This study sheds light on the complex mechanisms of bacterial ROS defense and offers FadR as a novel target for ROS control research.

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans.

Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.

Theoretical study of the nonlinear magnon-phonon coupling in CoF2.

The coupling and interplay between magnon and phonon are important topics for spintronics and magnonics. In this work we studied the nonlinear magnon-phonon coupling in CoF2. First-principles calculations demonstrate that the antiferromagnetic resonance magnon drives a phonon with B1gcharacter; the oscillating driving force has a frequency twice of that of the magnon. Comparing with similar materials indicates a strong correlation between the strength of nonlinear magnon-phonon coupling and the orbital magnetic moment of the magnetic ion. This work pave the way for theoretical study of nonlinear magnon-phonon coupling.

ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates the Warburg effect in breast cancer.

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked ß-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.

Effect of terahertz waves on the aggregation behavior of neurotransmitters.

Understanding the dynamics of neurotransmitters is crucial for unraveling synaptic transmission mechanisms in neuroscience. In this study, we investigated the impact of terahertz (THz) waves on the aggregation of four common neurotransmitters through all-atom molecular dynamics (MD) simulations. The simulations revealed enhanced nicotine (NCT) aggregation under 11.05 and 21.44 THz, with a minimal effect at 42.55 THz. Structural analysis further indicated strengthened intermolecular interactions and weakened hydration effects under specific THz stimulation. In addition, enhanced aggregation was observed at stronger field strengths, particularly at 21.44 THz. Furthermore, similar investigations on epinephrine (EPI), 5-hydroxytryptamine (5-HT), and γ-aminobutyric acid (GABA) corroborated these findings. Notably, EPI showed increased aggregation at 19.05 THz, emphasizing the influence of vibrational modes on aggregation. However, 5-HT and GABA, with charged or hydrophilic functional groups, exhibited minimal aggregation under THz stimulation. The present study sheds some light on neurotransmitter responses to THz waves, offering implications for neuroscience and interdisciplinary applications.

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

Specific molecular weight of Lycium barbarum polysaccharide for robust breast cancer regression by repolarizing tumor-associated macrophages.

The pro-tumorigenic M2-type tumor-associated macrophages (TAMs) in the immunosuppressive tumor microenvironment (TME) promote the progression, angiogenesis, and metastasis of breast cancer. The repolarization of TAMs from an M2-type toward an M1-type holds great potential for the inhibition of breast cancer. Here, we report that Lycium barbarum polysaccharides (LBPs) can significantly reconstruct the TME by modulating the function of TAMs. Specifically, we separated four distinct molecular weight segments of LBPs and compared their repolarization effects on TAMs in TME. The results showed that LBP segments within 50-100 kDa molecular weight range exhibited the prime effect on the macrophage repolarization, augmented phagocytosis effect of the repolarized macrophages on breast cancer cells, and regression of breast tumor in a tumor-bearing mouse model. In addition, RNA-sequencing confirms that this segment of LBP displays an enhanced anti-breast cancer effect through innate immune responses. This study highlights the therapeutic potential of LBP segments within the 50-100 kDa molecular weight range for macrophage repolarization, paving ways to offer new strategies for the treatment of breast cancer.

Effect of Mechanical Ventilation Mode Type on Postoperative Pulmonary Complications After Cardiac Surgery: A Randomized Controlled Trial.

OBJECTIVES: It is unknown whether there is a difference in pulmonary outcome in different intraoperative ventilation modes for cardiac surgery with cardiopulmonary bypass (CPB). The aim of this trial was to determine whether patients undergoing cardiac surgery with CPB could benefit from intraoperative optimal ventilation mode. DESIGN: This was a single-center, prospective, randomized controlled trial. SETTING: The study was conducted at a single-center tertiary-care hospital. PARTICIPANTS: A total of 1,364 adults undergoing cardiac surgery with CPB participated in this trial. INTERVENTIONS: Patients were assigned randomly (1:1:1) to receive 1 of 3 ventilation modes: volume-controlled ventilation (VCV), pressure-controlled ventilation (PCV), and pressure-controlled ventilation-volume guaranteed (PCV-VG). All arms of the study received the lung-protective ventilation strategy. MEASUREMENTS AND MAIN RESULTS: The primary outcome was a composite of postoperative pulmonary complications (PPCs) within the first 7 postoperative days. Pulmonary complications occurred in 168 of 455 patients (36.9%) in the PCV-VG group, 171 (37.6%) in the PCV group, and 182 (40.1%) in the VCV group, respectively. There was no statistical difference in the risk of overall pulmonary complications among groups (p = 0.585). There were no significant differences in the severity grade of PPCs within 7 days, postoperative ventilation duration, intensive care unit stay, postoperative hospital stay, or 30-day postoperative mortality. CONCLUSIONS: Among patients scheduled for cardiac surgery with CPB, intraoperative ventilation mode type did not affect the risk of postoperative pulmonary complications.

Test-Time Training for Deep MS/MS Spectrum Prediction Improves Peptide Identification.

In bottom-up proteomics, peptide-spectrum matching is critical for peptide and protein identification. Recently, deep learning models have been used to predict tandem mass spectra of peptides, enabling the calculation of similarity scores between the predicted and experimental spectra for peptide-spectrum matching. These models follow the supervised learning paradigm, which trains a general model using paired peptides and spectra from standard data sets and directly employs the model on experimental data. However, this approach can lead to inaccurate predictions due to differences between the training data and the experimental data, such as sample types, enzyme specificity, and instrument calibration. To tackle this problem, we developed a test-time training paradigm that adapts the pretrained model to generate experimental data-specific models, namely, PepT3. PepT3 yields a 10-40% increase in peptide identification depending on the variability in training and experimental data. Intriguingly, when applied to a patient-derived immunopeptidomic sample, PepT3 increases the identification of tumor-specific immunopeptide candidates by 60%. Two-thirds of the newly identified candidates are predicted to bind to the patient's human leukocyte antigen isoforms. To facilitate access of the model and all the results, we have archived all the intermediate files in Zenodo.org with identifier 8231084.

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition.

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through liquid-liquid phase separation. Here, we find that the phase separation of Caenorhabditis elegans SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation. We identify the SAD-1 kinase as a regulator of SYD-2 phase separation and determine presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. Using phosphoproteomics, we find SAD-1 phosphorylates SYD-2 on 3 sites that are critical to activate phase separation. Mechanistically, SAD-1 phosphorylation relieves a binding interaction between 2 folded domains in SYD-2 that inhibits phase separation by an intrinsically disordered region (IDR). We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, activating its phase separation and active zone assembly.

Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of fission yeast wtf meiotic drivers.

Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation. Here we investigate how the toxin and antidote products of a wtf-family KMD gene can act antagonistically. Both the toxin and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs PY motifs (Leu/Pro-Pro-X-Tyr) in its N-terminal cytosolic tail to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby preventing it from causing toxicity. A physical interaction between the antidote and the toxin enables the ubiquitinated antidote to translocate the toxin to the endosome and neutralize its toxicity. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs.

Effect of substituting on the transition dipole moment of the double perovskite Cs2AgInCl6.

The all-inorganic double perovskite Cs2AgInCl6with three dimensional structure has attracted much attention due to its direct bandgap property and particular luminescence mechanism, which is self-trapped exciton emission. However, it is a pity that Cs2AgInCl6exhibits low photoluminescence quantum yield, which affects its application for light-emitting devices. In this paper, the band structure and transition dipole moment of Cs2AgIn(1-x)SbxCl6(x= 0, 0.25, 0.5, 0.75) are calculated using first principle calculation. The calculated results shows that the pure material Cs2AgInCl6not only has a large band gap but also has the dipole forbidden transition, which means that the electrons cannot be excited from the valence band maximum to the conduction band minimum. However, the substituted Cs2AgIn0.75Sb0.25Cl6have a good property for the band gap about 3.066 eV and break forbidden transition at point X. The reason for its change is due to the overlap of electron and hole for charge density. Our work provides theoretical guidance for the design of more efficient light-emitting devices.

The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.