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Summary: In addition to the degradation of cell-cycle proteins, short-lived, damaged, or unfolded proteins are constantly cleared from cells by the proteasome. During proliferation, the proteasome localizes to the nucleus and cytoplasm; however, the functional relevance of this compartmentalization remains unclear. Here, we show that folding stress increases 26S/30S proteasome activity, which correlates with the upregulation of Ump1, a chaperone involved in 20S assembly. Conversely, ump1 inactivation results in a drop of 20S and 26S/30S proteasomes. Limited 26S/30S proteasomes in ump1-deficient cells accumulate in the nucleus where they degrade mitotic substrates, allowing cells to proceed through mitosis; however, these cells present cytoplasmic aggregates and constitutive activation of the heat shock response. Thus, our data suggest that an increase in proteasome assembly induced by folding stress functions as an additional layer to proteasome regulation and highlight the importance of balanced proteasome compartmentalization to sustain cell proliferation while maintaining proper cytoplasmic proteostasis.

Summary: Skeletal muscle cells (myofibers) are elongated non-mitotic, multinucleated syncytia that have adapted a microtubule lattice. Microtubule-associated proteins (MAPs) play roles in regulating microtubule architecture. The most abundant MAP in skeletal muscle is MAP4. MAP4 consists of a ubiquitous MAP4 isoform (uMAP4), expressed in most tissues, and a striated-muscle-specific alternatively spliced isoform (mMAP4) that includes a 3,180-nucleotide exon (exon 8). To determine the role of mMAP4 in skeletal muscle, we generated mice that lack mMAP4 and express only uMAP4 due to genomic deletion of exon 8. We demonstrate that loss of mMAP4 leads to disorganized microtubule architecture and intrinsic loss of force generation. We show that mMAP4 exhibits enhanced association with microtubules compared to uMAP4 and that both the loss of mMAP4 and the concomitant gain of uMAP4 cause loss of muscle function. These results demonstrate the critical role for balanced expression of mMAP4 and uMAP4 for skeletal muscle homeostasis.

Summary: P16INK4A expression is inversely associated with RB1 expression in cancer cells, and P16INK4A inhibits CDK4-catalyzed RB1 phosphorylation. How P16INK4A and RB1 coordinately express and regulate the cell cycle remains to be studied. In the present study, we found that P16INK4A upregulated the E3 ligase UTP14A, which led to the ubiquitination of RB1 at K810 and RB1 degradation. P16INK4A loss consistently disrupted the UTP14A-mediated degradation of RB1 and caused RB1 accumulation. Functionally, P16INK4A loss inhibited RB1 ubiquitination in a cell cycle progression-independent fashion and inhibited proteome-scale ubiquitination in a cell cycle progression-dependent manner. Our findings indicate that there is a negative feedback loop between P16INK4A and RB1 expression and that disruption of this loop may partially rescue the biological outcomes of P16INK4A loss. We also revealed a hitherto unknown function for P16INK4A in regulating proteome-scale ubiquitination by inhibiting cell proliferation, which may be useful for the development of anticancer drugs.

Summary: Cellular quiescence is a reversible and tightly regulated stem cell function essential for healthy aging. However, the elements that control quiescence during aging remain poorly defined. Using melanocyte stem cells (McSCs), we find that stem cell quiescence is neither passive nor static. For example, gene expression profiling of the transition from proliferating melanoblasts to quiescent melanocyte stem cells reveals tissue-specific regulation of the immune checkpoint protein PD-L1. In vitro, quiescence assays demonstrate that PD-L1 expression is a physiological attribute of quiescence in this cell lineage and reinforces this cell state. In vivo, a subset of quiescent McSCs is marked by PD-L1. While the overall number of McSCs decreases with age, PD-L1+ McSCs appear resistant to depletion. This phenomenon coincides with an aged McSC pool that exhibits a deeper transcriptomic quiescence. We predict that quiescent PD-L1+ stem cells retained with age may serve as cellular targets for reactivation.

Summary: Cystic fibrosis (CF) is a genetic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite reports of CFTR expression on endothelial cells, pulmonary vascular perturbations, and perfusion deficits in CF patients, the mechanism of pulmonary vascular disease in CF remains unclear. Here, our pilot study of 40 CF patients reveals a loss of small pulmonary blood vessels in patients with severe lung disease. Using a vessel-on-a-chip model, we establish a shear-stress-dependent mechanism of endothelial barrier failure in CF involving TRPV4, a mechanosensitive channel. Furthermore, we demonstrate that CFTR deficiency downregulates the function of PIEZO1, another mechanosensitive channel involved in angiogenesis and wound repair, and exacerbates loss of small pulmonary blood vessel. We also show that CFTR directly interacts with PIEZO1 and enhances its function. Our study identifies key cellular targets to mitigate loss of small pulmonary blood vessels in CF.

Summary: In response to corneal injury, an activation of corneal epithelial stem cells and their direct progeny the early transit amplifying (eTA) cells to rapidly proliferate is critical for proper re-epithelialization. Thus, it is important to understand how such stem/eTA cell activation is regulated. Angiotensin-converting enzyme 2 (ACE2) is predominantly expressed in the stem/eTA-enriched limbal epithelium but its role in the limbal epithelium was unclear. Single cell RNA sequencing (scRNA-seq) suggested that Ace2 involved the proliferation of the stem/eTA cells. Ace2 was reduced following corneal injury. Such reduction enhanced limbal epithelial proliferation and downregulated LCN2, a negative regulator of proliferation in a variety of tissues, via upregulating TGFA and consequently activating epidermal growth factor receptor (EGFR). Inhibition of EGFR or overexpression of LCN2 reversed the increased proliferation in limbal epithelial cells lacking ACE2. Our findings demonstrate that after corneal injury, ACE2 is downregulated, which activates limbal epithelial cell proliferation via a TGFA/EGFR/LCN2 pathway.

Summary: Rod-shaped fission yeast grows through cell wall expansion at poles and septum, synthesized by essential glucan synthases. Bgs1 synthesizes the linear β(1,3)glucan of primary septum at cytokinesis. Linear β(1,3)glucan is also present in the wall poles, suggesting additional Bgs1 roles in growth polarity. Our study reveals an essential collaboration between Bgs1 and Tea1-Tea4, but not other polarity factors, in controlling growth polarity. Simultaneous absence of Bgs1 function and Tea1-Tea4 causes complete loss of growth polarity, spread of other glucan synthases, and spherical cell formation, indicating this defect is specifically due to linear β(1,3)glucan absence. Furthermore, linear β(1,3)glucan absence induces actin patches delocalization and sterols spread, which are ultimately responsible for the growth polarity loss without Tea1-Tea4. This suggests strong similarities in Bgs1 functions controlling actin structures during cytokinesis and polarized growth. Collectively, our findings unveil that cell wall β(1,3)glucan regulates polarized growth, like the equivalent extracellular matrix in neuronal cells.

Summary: Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and myeloid skewing. We report that culture of bone marrow (BM) HSCs from aged mice with epidermal growth factor (EGF) suppressed myeloid skewing, increased multipotent colony formation, and increased HSC repopulation in primary and secondary transplantation assays. Mice transplanted with aged, EGF-treated HSCs displayed increased donor cell engraftment within BM HSCs and systemic administration of EGF to aged mice increased HSC self-renewal capacity in primary and secondary transplantation assays. Expression of a dominant negative EGFR in Scl/Tal1+ hematopoietic cells caused increased myeloid skewing and depletion of long term-HSCs in 15-month-old mice. EGF treatment decreased DNA damage in aged HSCs and shifted the transcriptome of aged HSCs from genes regulating cell death to genes involved in HSC self-renewal and DNA repair but had no effect on HSC senescence. These data suggest that EGFR signaling regulates the repopulating capacity of aged HSCs.

Summary: Stress granules (SGs) are membrane-less cellular compartments which are dynamically assembled via biomolecular condensation mechanism when eukaryotic cells encounter environmental stresses. SGs are important for gene expression and cell fate regulation. Dysregulation of SG homeostasis has been linked to human neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we report that the HRD1-SEL1L ubiquitin ligase complex specifically regulates the homeostasis of heat shock-induced SGs through the ubiquitin-proteasome system (UPS) and the UPS-associated ATPase p97. Mechanistically, the HRD1-SEL1L complex mediates SG homeostasis through the BiP-coupled PERK-eIF2α signaling axis of endoplasmic reticulum (ER) stress, thereby coordinating the unfolded protein response (UPR) with SG dynamics. Furthermore, we show that the distinctive branches of ER stress play differential roles in SG homeostasis. Our study indicates that the UPS and the UPR together via the HRD1-SEL1L ubiquitin ligase to maintain SG homeostasis in a stressor-dependent manner.

Summary: Multiple metabolic events occur in mitochondria. Mitochondrial protein translocation from the cytoplasm across compartments depends on the amino acid sequence within the precursor. At the mitochondria associated-ER membrane, misfolding of a mitochondrial targeted protein prior to import ablates metabolism. CYP11A1, cytochrome P450 cholesterol side chain cleavage enzyme (SCC), is imported from the cytoplasm to mitochondrial matrix catalyzing cholesterol to pregnenolone, an essential step for metabolic processes and mammalian survival. Multiple steps regulate the availability of an actively folded SCC; however, the mechanism is unknown. We identified that a dry molten globule state of SCC exists in the matrix by capturing intermediate protein folding steps dictated by its C-terminus. The intermediate dry molten globule state in the mitochondrial matrix of living cells is stable with a limited network of interaction and is inactive. The dry molten globule is activated with hydrogen ions availability, triggering cleavage of cholesterol sidechain, and initiating steroidogenesis.

Summary: Our work aimed to investigate the interactive roles of transforming growth factor β1 (TGF-β1), ubiquitin-specific-processing protease 7 (USP7), and Yes-associated protein (YAP) in ferroptosis during sepsis-secondary acute lung injury (ALI). Our study demonstrated that ferroptosis was aggravated by TGF-β1 in both cellular and animal models of acute lung injury. Additionally, YAP upregulated glutathione peroxidase 4 (GPX4) and SLC7A11 by regulating the binding of TEAD4 to GPX4/SLC7A11 promoters. Furthermore, large tumor suppressor kinase 1 (LATS1) knockdown resulted in YAP expression stimulation, while USP7 downregulated YAP via deubiquitinating and stabilizing LATS1/2. YAP overexpression or USP7/LATS1 silencing reduced ferroptosis process, which regulated YAP through a feedback loop. However, TGF-β1 annulled the repression of ferroptosis by YAP overexpression or LATS1/USP7 knockdown. By elucidating the molecular interactions between TGF-β1, USP7, LATS1/2, and YAP, we identified a new regulatory axis of ferroptosis in sepsis-secondary ALI. Our study sheds light on the pathophysiology of ferroptosis and proposes a potential therapeutic approach for sepsis-induced ALI.

Summary: The efficacy of T cell therapies in treating solid tumors is limited by poor in vivo persistence, proliferation, and cytotoxicity, which can be attributed to limited and variable ex vivo activation. Herein, we present a 10-day kinetic profile of T cells subjected to fluid shear stress (FSS) ex vivo, with and without stimulation utilizing bead-conjugated anti-CD3/CD28 antibodies. We demonstrate that mechanical stimulation via FSS combined with bead-bound anti-CD3/CD28 antibodies yields a synergistic effect, resulting in amplified and sustained downstream signaling (NF-κB, c-Fos, and NFAT), expression of activation markers (CD69 and CD25), proliferation and production of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2). This study represents the first characterization of the dynamic response of primary T cells to FSS. Collectively, our findings underscore the critical role of mechanosensitive ion channel-mediated mechanobiological signaling in T cell activation and fitness, enabling the development of strategies to address the current challenges associated with poor immunotherapy outcomes.

Summary: Muscle contraction is vital for animal survival, and the sarcomere is the fundamental unit for this process. However, the functions of many conserved sarcomere proteins remain unknown, as their mutants do not exhibit obvious defects. To address this, Caenorhabditis elegans was utilized as a model organism to investigate RSU-1 function in the body wall muscle. RSU-1 is found to colocalize with UNC-97 at the dense body and M-line, and it is particularly crucial for regulating locomotion in aging worms, rather than in young worms. This suggests that RSU-1 has a specific function in maintaining muscle function during aging. Furthermore, the interaction between RSU-1 and UNC-97/PINCH is essential for RSU-1 to modulate locomotion, preserve filament structure, and sustain the M-line and dense body throughout aging. Overall, these findings highlight the significant contribution of RSU-1, through its interaction with UNC-97, in maintaining proper muscle cell function in aging worms.

Summary: The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico-to-in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions.

Summary: Mitochondrial dynamics is a process that balances fusion and fission events, the latter providing a mechanism for segregating dysfunctional mitochondria. Fission is controlled by the mitochondrial membrane potential (ΔΨm), optic atrophy 1 (OPA1) cleavage, and DRP1 recruitment. It is thought that this process is closely linked to the activity of the mitochondrial respiratory chain (MRC). However, we report here that MRC inhibition does not decrease ΔΨm nor increase fission, as evidenced by hyperconnected mitochondria. Conversely, blocking F0F1-ATP synthase activity induces fragmentation. We show that the F0F1-ATP synthase is sensing the inhibition of MRC activity by immediately promoting its reverse mode of action to hydrolyze matrix ATP and restoring ΔΨm, thus preventing fission. While this reverse mode is expected to be inhibited by the ATPase inhibitor ATPIF1, we show that this sensing is independent of this factor. We have unraveled an unexpected role of F0F1-ATP synthase in controlling the induction of fission by sensing and maintaining ΔΨm.

Summary: Lysosomes, the hub of metabolic signaling, are associated with various diseases and participate in autophagy by supplying nutrients to cells under nutrient starvation. However, their function and regulation under glucose starvation remain unclear and are studied herein. Under glucose starvation, lysosomal protein expression decreased, leading to the accumulation of damaged lysosomes. Subsequently, cell death occurred via ferroptosis and iron accumulation due to DMT1 degradation. GPX4, a key factor in ferroptosis inhibition located on the outer membrane of lysosomes, accumulated in lysosomes, especially under glucose starvation, to protect cells from ferroptosis. ALDOA, GAPDH, NAMPT, and PGK1 are also located on the outer membrane of lysosomes and participate in lysosomal function. These enzymes did not function effectively under glucose starvation, leading to lysosomal dysfunction and ferroptosis. These findings may facilitate the treatment of lysosomal-related diseases.

Summary: It is imperative to explore biomarkers that are both precise and readily accessible in the comprehensive management of breast cancer. A multicenter cohort, including 512 breast cancer patients and 198 nonneoplastic individuals, was recruited to detect the level of tumor-derived extracellular vesicles using our method based on dual DNA tetrahedral nanostructures. The level of tumor-derived extracellular vesicles was significantly higher in newly diagnosed breast cancer patients than in nonneoplastic individuals at a cutoff value of 3.58 U/μL. For postoperative metastasis monitoring, the level of tumor-derived extracellular vesicles was significantly higher in breast cancer patients with metastasis than in those without metastasis at a cutoff value of 3.91 U/μL. Its efficacy of diagnosis and metastasis monitoring was superior to traditional tumor markers. Elevated level of tumor-derived extracellular vesicles served as a predictive biomarker for diagnosis and metastasis monitoring in breast cancer patients.

Summary: Glia are the protectors of the nervous system, providing neurons with support and protection from cytotoxic insults. We previously discovered that four astrocyte-like glia can regulate organismal proteostasis and longevity in C. elegans. Expression of the UPRER transcription factor, XBP-1s, in these glia increases stress resistance, and longevity, and activates the UPRER in intestinal cells via neuropeptides. Autophagy, a key regulator of metabolism and aging, has been described as a cell autonomous process. Surprisingly, we find that glial XBP-1s enhances proteostasis and longevity by cell non-autonomously reprogramming organismal lipid metabolism and activating autophagy. Glial XBP-1s regulates the activation of another transcription factor, HLH-30/TFEB, in the intestine. HLH-30 activates intestinal autophagy, increases intestinal lipid catabolism, and upregulates a robust transcriptional program. Our study reveals a novel role for glia in regulating peripheral lipid metabolism, autophagy, and organellar health through peripheral activation of HLH-30 and autophagy.

Summary: Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella’s virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Summary: O-GlcNAc transferase (OGT) acts in the development of various cancers, but its role in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, we found that OGT was upregulated in ccRCC and this upregulation was associated with a worse survival. Moreover, OGT promoted the proliferation, clone formation, and invasion of VHL-mutated ccRCC cells. Mechanistically, OGT increased the protein level of hypoxia-inducible factor-2α (HIF-2α) (the main driver of the clear cell phenotype) by repressing ubiquitin‒proteasome system-mediated degradation. Interestingly, the OGT/HIF-2α axis conferred ccRCC a high sensitivity to ferroptosis. In conclusion, OGT promotes the progression of VHL-mutated ccRCC by inhibiting the degradation of HIF-2α, and agents that can modulate the OGT/HIF-2α axis may exert therapeutic effects on mutated VHL ccRCC.

Summary: G protein-coupled receptors (GPCRs) constitute the largest superfamily of plasma membrane signaling proteins. However, virtually nothing is known about their recruitment to COPII vesicles for forward delivery after synthesis in the endoplasmic reticulum (ER). Here, we demonstrate that some GPCRs are highly concentrated at ER exit sites (ERES) before COPII budding. Angiotensin II type 2 receptor (AT2R) and CXCR4 concentration are directed by a di-acidic motif and a 9-residue domain, respectively, and these motifs also control receptor ER-Golgi traffic. We further show that AT2R interacts with Sar1 GTPase and that distinct GPCRs have different ER-Golgi transport rates via COPII which is independent of their concentration at ERES. Collectively, these data demonstrate that GPCRs can be actively captured by COPII via specific motifs and direct interaction with COPII components that in turn affects their export dynamics, and provide important insights into COPII targeting and forward trafficking of nascent GPCRs.

Summary: Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 “WY” conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 μM binding affinity for CtUGGTGT24 in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Summary: Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high-affinity binding, namely more than eight base pairs, with the 5′SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5′SS base-pairing or other regulatory obstructions. Here, we show in vitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5′SS, and hence promotes the inclusion of exons containing 5′SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing.

Summary: Bone stress injuries are common overuse injuries, especially in soldiers, athletes, and performers. In contrast to various post-injury treatments, early protection against bone stress injuries can provide greater benefit. This study explored the early protection strategies against bone stress injuries by mobilization of endogenous targeted bone remodeling. The effects of various pharmaceutical/biophysical approaches, individual or combinational, were investigated by giving intervention before fatigue loading. We optimized the dosage and administration parameters and found that early intervention with pulsed electromagnetic field and parathyroid hormone (i.e., PEMF+PTH) resulted in the most pronounced protective effects among all the approaches against the bone stress injuries. In addition, the mechanisms by which the strategy mobilizes targeted bone remodeling and enhances the self-repair capacity of bone were systematically investigated. This study proposes strategies to reduce the incidence of bone stress injuries in high-risk populations (e.g., soldiers and athletes), particularly for those before sudden increased physical training.

Summary: We present a study on the nuclear export efficiency and time of pre-ribosomal subunits in live mammalian cells, using high-speed single-molecule tracking and single-molecule fluorescence resonance energy transfer techniques. Our findings reveal that pre-ribosomal particles exhibit significantly higher nuclear export efficiency compared to other large cargos like mRNAs, with around two-thirds of interactions between the pre-60S or pre-40S and the nuclear pore complexes (NPCs) resulting in successful export to the cytoplasm. We also demonstrate that nuclear transport receptor (NTR) chromosomal maintenance 1 (CRM1) plays a crucial role in nuclear export efficiency, with pre-60S and pre-40S particle export efficiency decreasing by 11–17-fold when CRM1 is inhibited. Our results suggest that multiple copies of CRM1 work cooperatively to chaperone pre-ribosomal subunits through the NPC, thus increasing export efficiency and decreasing export time. Significantly, this cooperative NTR mechanism extends beyond pre-ribosomal subunits, as evidenced by the enhanced nucleocytoplasmic transport of proteins.

Summary: Several heat shock proteins are implicated in the endogenous cardioprotective mechanisms, but little is known about the role of heat shock protein beta-1 (HSPB1). This study aims to investigate the oxidation state and role of HSPB1 in cardiomyocytes undergoing oxidative stress and underlying mechanisms. Here, we demonstrate that hydrogen peroxide (H2O2) promotes the homo-oxidation of HSPB1. Cys137 residue of HSPB1 is not only required for it to protect cardiomyocytes against oxidative injury but also modulates its oxidation, phosphorylation at Ser15, and distribution to insoluble cell components after H2O2 treatment. Moreover, Cys137 residue is indispensable for HSPB1 to interact with KEAP1, thus regulating its oxidation and intracellular distribution, subsequently promoting the nuclear translocation of NRF2, and increasing the transcription of GLCM, HMOX1, and TXNRD1. Altogether, these findings provide evidence that Cys137 residue is indispensable for HSPB1 to maintain its redox state and antioxidant activity via activating KEAP1/NRF2 signaling cascade in cardiomyocytes.

Summary: Acute myeloid leukemia (AML) is the type of hematologic neoplasm most common in adults. Glucocorticoid-induced gene TSC22D3 regulates cell proliferation through its function as a transcription factor. However, there is no consensus on the prognostic and immunoregulatory significance of TSC22D3 in AML. In the present study, we evaluated the correlation between TSC22D3 expression, immunoinfiltration, and prognostic significance in AML. Knockdown of TSC22D3 significantly attenuated the proliferation of Hel cells and increased sensitivity to cytarabine (Ara-c) drugs. Furthermore, TSC22D3 reduced the release of interleukin-1β (IL-1β) by inhibiting the NF-κB/NLRP3 signaling pathway, thereby inhibiting macrophage polarization to M1 subtype, and attenuating the pro-inflammatory tumor microenvironment. In conclusion, this study identified TSC22D3 as an immune-related prognostic biomarker for AML patients and suggested that therapeutic targeting of TSC22D3 may be a potential treatment option for AML through tumor immune escape.

Summary: Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass.

Summary: The FliG protein plays a pivotal role in switching the rotational direction of the flagellar motor between clockwise and counterclockwise. Although we previously showed that mutations in the Gly-Gly linker of FliG induce a defect in switching rotational direction, the detailed molecular mechanism was not elucidated. Here, we studied the structural changes in the FliG fragment containing the middle and C-terminal regions, named FliGMC, and the switch-defective FliGMC-G215A, using nuclear magnetic resonance (NMR) and molecular dynamics simulations. NMR analysis revealed multiple conformations of FliGMC, and the exchange process between these conformations was suppressed by the G215A residue substitution. Furthermore, changes in the intradomain orientation of FliG were induced by changes in hydrophobic interaction networks throughout FliG. Our finding applies to FliG in a ring complex in the flagellar basal body, and clarifies the switching mechanism of the flagellar motor.

Summary: Reactive oxygen species (ROS), such as superoxide, are crucial factors involved in the stimulation of cellular aging. Mitochondria, which are important organelles responsible for various metabolic processes in cells, produce ROS. These ROS impair mitochondrial function, thereby accelerating aging-related cellular dysfunction. Herein, we demonstrated that the Spirulina polysaccharide complex (SPC) restores mitochondrial function and collagen production by scavenging superoxide via the upregulation of superoxide dismutase 2 (SOD2) in aging fibroblasts. We observed that SOD2 expression was linked to inflammatory pathways; however, SPC did not upregulate the expression of most inflammatory cytokines produced as a result of induction of LPS in aging fibroblasts, indicating that SPC induces SOD2 without activation of inflammatory pathways. Furthermore, SPC stimulated endoplasmic reticulum (ER) protein folding by upregulating ER chaperones expression. Thus, SPC is proposed to be an antiaging material that rejuvenates aging fibroblasts by increasing their antioxidant potential via the upregulation of SOD2.

Summary: TRIM24 is an oncogenic chromatin reader that is frequently overexpressed in human tumors and associated with poor prognosis. However, TRIM24 is rarely mutated, duplicated, or rearranged in cancer. This raises questions about how TRIM24 is regulated and what changes in its regulation are responsible for its overexpression. Here, we perform a genome-wide CRISPR-Cas9 screen by fluorescence-activated cell sorting (FACS) that nominated 220 negative regulators and elucidated a regulatory network that includes the KAP1 corepressor, CNOT deadenylase, and GID/CTLH E3 ligase. Knocking out required components of these three complexes caused TRIM24 overexpression, confirming their negative regulation of TRIM24. Our findings identify regulators of TRIM24 that nominate previously unexplored contexts for this oncoprotein in biology and disease. These findings were enabled by SLIDER, a new scoring system designed and vetted in our study as a broadly applicable tool for analysis of CRISPR screens performed by FACS.

Summary: Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all “cancer genes” were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: (1) proximity of genes able to produce prematurely terminated transcripts, which lead to the production of (2) trans-spliced fusion RNAs, and finally, the induction of (3) DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced. The implications of these findings will be discussed.

Summary: Osteoclasts are specialized multinucleated giant cells with unique bone-destroying capacities. A recent study revealed that osteoclasts undergo an alternative cell fate by dividing into daughter cells called osteomorphs. To date, no studies have focused on the mechanisms of osteoclast fission. In this study, we analyzed the alternative cell fate process in vitro and, herein, reported the high expression of mitophagy-related proteins during osteoclast fission. Mitophagy was further confirmed by the colocalization of mitochondria with lysosomes, as observed in fluorescence images and transmission electron microscopy. We investigated the role played by mitophagy in osteoclast fission via drug stimulation experiments. The results showed that mitophagy promoted osteoclast division, and inhibition of mitophagy induced osteoclast apoptosis. In summary, this study reveals the role played by mitophagy as the decisive link in osteoclasts’ fate, providing a new therapeutic target and perspective for the clinical treatment of osteoclast-related diseases.

Summary: Creatine kinase (CK) is an essential metabolic enzyme mediating creatine/phosphocreatine interconversion and shuttle to replenish ATP for energy needs. Ablation of CK causes a deficiency in energy supply that eventually results in reduced muscle burst activity and neurological disorders in mice. Besides the well-established role of CK in energy-buffering, the mechanism underlying the non-metabolic function of CK is poorly understood. Here we demonstrate that creatine kinase brain-type (CKB) may function as a protein kinase to regulate BCAR1 Y327 phosphorylation that enhances the association between BCAR1 and RBBP4. Then the complex of BCAR1 and RPPB4 binds to the promoter region of DNA damage repair gene RAD51 and activates its transcription by modulating histone H4K16 acetylation to ultimately promote DNA damage repair. These findings reveal the possible role of CKB independently of its metabolic function and depict the potential pathway of CKB-BCAR1-RBBP4 operating in DNA damage repair.

Summary: The COP9 signalosome (CSN) and cullin-RING ubiquitin ligases (CRLs) form latent CSN-CRL complexes detectable in cells. We demonstrate that the CSN variants CSNCSN7A and CSNCSN7B preferentially bind to CRL3 or CRL4A and CRL4B, respectively. Interestingly, the interacting protein ubiquitin-specific protease 15 exclusively binds to latent CSNCSN7A-CRL3, while p27KIP attaches to latent CSNCSN7B-CRL4A complex. Inhibition of deneddylation by CSN5i-3 or neddylation by MLN4924 do not impede the formation of latent complexes. Latent CSNCSN7A-CRL3 and latent CSNCSN7B-CRL4A/B particles are essential for specific cellular functions. We found that curcumin-induced cell death requires latent CSNCSN7B-CRL4A. Knockout of CSN7B in HeLa cells leads to resistance against curcumin. Remarkably, the small GTPase RAB18 recruits latent CSNCSN7A-CRL3 complex to lipid droplets (LDs), where CRL3 is activated by neddylation, an essential event for LD formation during adipogenesis. Knockdown of CSN7A or RAB18 or destabilization of latent complexes by cutting off CSN7A C-terminal 201–275 amino acids blocks adipogenesis.

Summary: Accurate spatiotemporal control of multicellular self-organization by various signaling pathways is essential for developmental stages. In particular, evolutionarily conserved Wnt signaling serves as a major morphogenetic switch to determine the anteroposterior axis of the embryo. Here, we developed a genetically encoded optochemogenetic Wnt switch, named optochemoWnt, by coupling a blue light-inducible CRY2olig and rapamycin-inducible LRP6c clustering. The rationally designed optochemoWnt successfully modulated Wnt signaling with AND-gated patterns and demonstrated an improved signal-to-noise ratio (SNR). The dual-triggered switch provides a safeguard to prevent signal leakage resulting from ambient light sources under general laboratory conditions. OptochemoWnt expands the molecular toolbox available for the fields of developmental biology and tissue engineering. In addition, the AND-gated strategy of optochemoWnt may be used for other biomedical applications that integrate user defined switch elements with Boolean logic gates.

Summary: The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.

Summary: Active filament networks can organize into various dynamic architectures driven by cross-linking motors. Densities and kinetic properties of motors and microtubules have been shown previously to determine active microtubule network self-organization, but the effects of other control parameters are less understood. Using computer simulations, we study here how microtubule lengths and crowding effects determine active network architecture and dynamics. We find that attractive interactions mimicking crowding effects or long microtubules both promote the formation of extensile nematic networks instead of asters. When microtubules are very long and the network is highly connected, a new isotropically motile network state resembling a “gliding mesh” is predicted. Using in vitro reconstitutions, we confirm the existence of this gliding mesh experimentally. These results provide a better understanding of how active microtubule network organization can be controlled, with implications for cell biology and active materials in general.

Summary: PTEN encodes a tumor suppressor with lipid and protein phosphatase activities whose dysfunction has been implicated in melanomagenesis; less is known about how its phosphatases regulate melanoma metastasis. We demonstrate that PTEN expression negatively correlates with metastatic progression in human melanoma samples and a PTEN-deficient mouse melanoma model. Wildtype PTEN expression inhibited melanoma cell invasiveness and metastasis in a dose-dependent manner, behaviors that specifically required PTEN protein phosphatase activity. PTEN phosphatase activity regulated metastasis through Entpd5. Entpd5 knockdown reduced metastasis and IGF1R levels while promoting ER stress. In contrast, Entpd5 overexpression promoted metastasis and enhanced IGF1R levels while reducing ER stress. Moreover, Entpd5 expression was regulated by the ER stress sensor ATF6. Altogether, our data indicate that PTEN phosphatase activity inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6, thereby identifying novel candidate therapeutic targets for the treatment of PTEN mutant melanoma.

Summary: Centrosomal protein 120 (CEP120) is a 120 kDa centrosome protein that plays an important role in centrosome replication. Overexpression of CEP120 can lead to centrosome duplicate abnormality, which is closely associated with tumorigenesis and development. However, there are no reports on the relationship between CEP120 and tumors. In our study, overexpression of CEP120 promoted centrosome amplification in gastric cancer (GC), and the role of CEP120 in promoting GC progression was demonstrated in vitro and in vivo. We demonstrated that CEP120 promotes centrosome amplification and GC progression by promoting the expression and centrosome aggregation of the deubiquitinating enzyme USP54, maintaining the stability of PLK4 and reducing its ubiquitination degradation. In conclusion, the CEP120-USP54-PLK4 axis may play an important role in promoting centrosome amplification and GC progression, thus providing a potential therapeutic target for GC.

Summary: Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Summary: Tumors with BRCA1 mutations have poor prognoses due to genomic instability. Yet this genomic instability has risks and BRCA1-deficient (def) cancer cells must develop pathways to mitigate these risks. One such risk is the accumulation of unfolded proteins in BRCA1-def cancers from increased mutations due to their loss of genomic integrity. Little is known about how BRCA1-def cancers survive their genomic instability. Here we show that BRCA1 is an E3 ligase in the endoplasmic reticulum (ER) that targets the unfolded protein response (UPR) stress sensors, Eukaryotic Translation Initiation Factor 2-alpha Kinase 3 (PERK) and Serine/Threonine-Protein Kinase/Endoribonuclease Inositol-Requiring Enzyme 1 (IRE1) for ubiquitination and subsequent proteasome-mediated degradation. When BRCA1 is mutated or depleted, both PERK and IRE1 protein levels are increased, resulting in a constitutively activated UPR. Furthermore, the inhibition of protein folding or UPR signaling markedly decreases the overall survival of BRCA1-def cancer cells. Our findings define a mechanism used by the BRCA1-def cancer cells to survive their increased unfolded protein burden which can be used to develop new therapeutic strategies to treat these cancers.

Summary: Mutations and defects in nuclear lamins can cause major pathologies, including inflammation and inflammatory diseases. Yet, the underlying molecular mechanisms are not known. We now report that the pro-inflammatory activation of macrophages, as induced by LPS or pathogenic E. coli, reduces Lamin-A/C levels thereby augmenting pro-inflammatory gene expression and cytokine secretion. We show that the activation of bone-marrow-derived macrophages (BMDMs) causes the phosphorylation and degradation of Lamin-A/C, as mediated by CDK1 and Caspase-6, respectively, necessary for upregulating IFN-β expression. Enhanced IFN-β expression subsequently increases pro-inflammatory gene expression via the IFN-β-STAT axis. Pro-inflammatory gene expression was also amplified in the complete absence of Lamin-A/C. Alternatively, pharmacological inhibition of either Lamin-A/C phosphorylation or degradation significantly downregulated pro-inflammatory gene expression, as did the targeting of IFN-β-STAT pathway members, i.e. phospho-STAT1 and phospho-STAT3. As Lamin-A/C is a previously unappreciated regulator of the pro-inflammatory macrophage response, our findings suggest novel opportunities to treat inflammatory diseases.

Summary: The machinery of intraflagellar transport (IFT) consists of IFT motors and the ciliary cargo adaptors including IFT-A and IFT-B complexes and BBSome. IFT-B, which is composed of IFT-B1 and IFT-B2 subcomplexes, interacts with IFT motors and IFT-A during anterograde IFT and IFT-A during retrograde IFT while it is also implicated in BBSome trafficking. However, the assembly and stability of IFT-B and its regulation of anterograde IFT and BBSome trafficking remain not clear. Here, we show that IFT38 functions in the regulation of anterograde IFT and retrograde trafficking of BBSome. Deletion of IFT-B1 or IFT-B2 subunits results in differential instability of IFT-B1 and IFT-B2. The stability of IFT-B1 and IFT-B2 is mutually dependent and mediated by the connecting tetramer IFT38/5788/52. The formation of an intact IFT-B1 and IFT-B2 is not altered by the deletion of IFT38 of IFT-B2 and IFT52 of IFT-B1, respectively. Further analysis suggests a modular pathway for IFT-B assembly.

Summary: Radiation therapy damages tumors and normal tissues, probably in part through the recruitment of immune cells. Endothelial high-mannose N-glycans are, in particular, involved in monocyte-endothelium interactions. Trimmed by the class I α-mannosidases, these structures are quite rare in normal conditions. Here, we show that the expression of the endothelial α-mannosidase MAN1C1 protein decreases after irradiation. We modeled two crucial steps in monocyte recruitment by developing in vitro real-time imaging models. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion and enhances transendothelial migration of monocytes. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium.

Summary: An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.

Summary: Skeletal muscle generation of ammonia, an endogenous cytotoxin, is increased during exercise. Perturbations in ammonia metabolism consistently occur in chronic diseases, and may blunt beneficial skeletal muscle molecular responses and protein homeostasis with exercise. Phosphorylation of skeletal muscle proteins mediates cellular signaling responses to hyperammonemia and exercise. Comparative bioinformatics and machine learning-based analyses of published and experimentally derived phosphoproteomics data identified differentially expressed phosphoproteins that were unique and shared between hyperammonemic murine myotubes and skeletal muscle from exercise models. Enriched processes identified in both hyperammonemic myotubes and muscle from exercise models with selected experimental validation included protein kinase A (PKA), calcium signaling, mitogen-activated protein kinase (MAPK) signaling, and protein homeostasis. Our approach of feature extraction from comparative untargeted “omics” data allows for selection of preclinical models that recapitulate specific human exercise responses and potentially optimize functional capacity and skeletal muscle protein homeostasis with exercise in chronic diseases.

Summary: Neurofibromatosis type 2 is an autosomal dominant multiple neoplasia syndrome and is usually caused by mutations in the neurofibromin 2 (NF2) gene, which encodes a tumor suppressor and initiates the Hippo pathway. However, the mechanism by which NF2 functions in the Hippo pathway isn’t fully understood. Here we identified a NF2 c.770-784del mutation from a neurofibromatosis type 2 family. MD simulations showed that this mutation significantly changed the structure of the F3 module of the NF2-FERM domain. Functional assays indicated that the NF2 c.770-784del variant formed LLPS in the cytoplasm with LATS to restrain LATS plasma membrane localization and inactivated the Hippo pathway. Besides, this deletion partly caused a skipping of exon 8 and reduced the protein level of NF2, collectively promoting proliferation and tumorigenesis of meningeal cells. We identified an unrecognized mechanism of LLPS and splicing skipping for the NF2-induced Hippo pathway, which provided new insight into the pathogenesis of neurofibromatosis type 2.

Summary: The ribonuclease A (RNase A) family is one of the best-characterized vertebrate-specific proteins. In humans, eight catalytically active RNases (numbered 1–8) have been identified and have unique tissue distributions. Apart from the digestion of dietary RNA, a broad range of biological actions, including the regulation of intra- or extra-cellular RNA metabolism as well as antiviral, antibacterial, and antifungal activities, neurotoxicity, promotion of cell proliferation, anti-apoptosis, and immunomodulatory abilities, have been recently reported for the members of this family. Based on multiple biological roles, RNases are found to participate in the pathogenic processes of many diseases, such as infection, immune dysfunction, neurodegeneration, cancer, and cardiovascular disorders. This review summarizes the available data on the human RNase A family and illustrates the significant roles of the eight canonical RNases in health and disease, for stimulating further basic research and development of ideas on the potential solutions for disease diagnosis and treatment.

Summary: Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.