Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy.

Autophagosome formation requires multiple autophagy-related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome-wide CRISPR screens identifying genes required for NBR1 flux in ATG7KO cells. We find that ATG7-independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1's ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin-independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor-mediated clustering of upstream autophagy factors drives continued autophagosome formation.

TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling.

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GßL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GßL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GßL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GßL to promote GßL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GßL, by either K305R/K313R mutations or a melanoma-associated GßL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GßL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.

Functional analysis of Cullin 3 E3 ligases in tumorigenesis.

Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.

Regulation of topoisomerase II stability and activity by ubiquitination and SUMOylation: clinical implications for cancer chemotherapy.

DNA topoisomerases II (TOP2) are peculiar enzymes (TOP2α and TOP2ß) that modulate the conformation of DNA by momentarily breaking double-stranded DNA to allow another strand to pass through, and then rejoins the DNA phosphodiester backbone. TOP2α and TOP2ß play vital roles in nearly all events involving DNA metabolism, including DNA transcription, replication, repair, and chromatin remodeling. Beyond these vital functions, TOP2 enzymes are therapeutic targets for various anticancer drugs, termed TOP2 poisons, such as teniposide, etoposide, and doxorubicin. These drugs exert their antitumor activity by inhibiting the activity of TOP2-DNA cleavage complexes (TOP2ccs) containing DNA double-strand breaks (DSBs), subsequently leading to the degradation of TOP2 by the 26S proteasome, thereby exposing the DSBs and eliciting a DNA damage response. Failure of the DSBs to be appropriately repaired leads to genomic instability. Due to this mechanism, patients treated with TOP2-based drugs have a high incidence of secondary malignancies and cardiotoxicity. While the cytotoxicity associated with TOP2 poisons appears to be TOP2α-dependent, the DNA sequence rearrangements and formation of DSBs appear to be mediated primarily through TOP2ß inhibition, likely due to the differential degradation patterns of TOP2α and TOP2ß. Research over the past few decades has shown that under various conditions, the ubiquitin-proteasome system (UPS) and the SUMOylation pathway are primarily responsible for regulating the stability and activity of TOP2 and are therefore critical regulators of the therapeutic effect of TOP2-targeting drugs. In this review, we summarize the current progress on the regulation of TOP2α and TOP2ß by ubiquitination and SUMOylation. By fully elucidating the basic biology of these essential and complex molecular mechanisms, better strategies may be developed to improve the therapeutic efficacy of TOP2 poisons and minimize the risks of therapy-related secondary malignancy.

Unraveling the Molecular Nexus between GPCRs, ERS, and EMT.

G protein-coupled receptors (GPCRs) represent a large family of transmembrane proteins that transduce an external stimulus into a variety of cellular responses. They play a critical role in various pathological conditions in humans, including cancer, by regulating a number of key processes involved in tumor formation and progression. The epithelial-mesenchymal transition (EMT) is a fundamental process in promoting cancer cell invasion and tumor dissemination leading to metastasis, an often intractable state of the disease. Uncontrolled proliferation and persistent metabolism of cancer cells also induce oxidative stress, hypoxia, and depletion of growth factors and nutrients. These disturbances lead to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and induce a cellular condition called ER stress (ERS) which is counteracted by activation of the unfolded protein response (UPR). Many GPCRs modulate ERS and UPR signaling via ERS sensors, IRE1α, PERK, and ATF6, to support cancer cell survival and inhibit cell death. By regulating downstream signaling pathways such as NF-κB, MAPK/ERK, PI3K/AKT, TGF-ß, and Wnt/ß-catenin, GPCRs also upregulate mesenchymal transcription factors including Snail, ZEB, and Twist superfamilies which regulate cell polarity, cytoskeleton remodeling, migration, and invasion. Likewise, ERS-induced UPR upregulates gene transcription and expression of proteins related to EMT enhancing tumor aggressiveness. Though GPCRs are attractive therapeutic targets in cancer biology, much less is known about their roles in regulating ERS and EMT. Here, we will discuss the interplay in GPCR-ERS linked to the EMT process of cancer cells, with a particular focus on oncogenes and molecular signaling pathways.

The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection.

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

Functional analyses of major cancer-related signaling pathways in Alzheimer's disease etiology.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease and accounts for majority of human dementia. The hyper-phosphorylated tau-mediated intracellular neurofibrillary tangle and amyloid ß-mediated extracellular senile plaque are characterized as major pathological lesions of AD. Different from the dysregulated growth control and ample genetic mutations associated with human cancers, AD displays damage and death of brain neurons in the absence of genomic alterations. Although various biological processes predominately governing tumorigenesis such as inflammation, metabolic alteration, oxidative stress and insulin resistance have been associated with AD genesis, the mechanistic connection of these biological processes and signaling pathways including mTOR, MAPK, SIRT, HIF, and the FOXO pathway controlling aging and the pathological lesions of AD are not well recapitulated. Hence, we performed a thorough review by summarizing the physiological roles of these key cancer-related signaling pathways in AD pathogenesis, comprising of the crosstalk of these pathways with neurofibrillary tangle and senile plaque formation to impact AD phenotypes. Importantly, the pharmaceutical investigations of anti-aging and AD relevant medications have also been highlighted. In summary, in this review, we discuss the potential role that cancer-related signaling pathways may play in governing the pathogenesis of AD, as well as their potential as future targeted strategies to delay or prevent aging-related diseases and combating AD.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

The intersection between aging and cardiovascular disease.

The average lifespan of humans is increasing, and with it the percentage of people entering the 65 and older age group is growing rapidly and will continue to do so in the next 20 years. Within this age group, cardiovascular disease will remain the leading cause of death, and the cost associated with treatment will continue to increase. Aging is an inevitable part of life and unfortunately poses the largest risk factor for cardiovascular disease. Although numerous studies in the cardiovascular field have considered both young and aged humans, there are still many unanswered questions as to how the genetic pathways that regulate aging in model organisms influence cardiovascular aging. Likewise, in the molecular biology of aging field, few studies fully assess the role of these aging pathways in cardiovascular health. Fortunately, this gap is beginning to close, and these two fields are merging together. We provide an overview of some of the key genes involved in regulating lifespan and health span, including sirtuins, AMP-activated protein kinase, mammalian target of rapamycin, and insulin-like growth factor 1 and their roles regulating cardiovascular health. We then discuss a series of review articles that will appear in succession and provide a more comprehensive analysis of studies carried out linking genes of aging and cardiovascular health, and perspectives of future directions of these two intimately linked fields.

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.

The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.

During autophagy, potentially toxic cargo is enveloped by a newly formed autophagosome and trafficked to the lysosome for degradation. Ubiquitinated protein aggregates, a key target for autophagy, are identified by multiple autophagy receptors. NBR1 is an archetypal autophagy receptor and an excellent model for deciphering the role of the multivalent, heterotypic interactions made by cargo-bound receptors. Using NBR1 as a model, we find that three critical binding partners - ATG8-family proteins, FIP200, and TAX1BP1 - each bind to a short linear interaction motif (SLiM) within NBR1. Mutational peptide arrays indicate that these binding events are mediated by distinct overlapping determinants, rather than a single, convergent, SLiM. AlphaFold modeling underlines the need for conformational flexibility within the NBR1 SLiM, as distinct conformations mediate each binding event. To test the extent to which overlapping SLiMs exist beyond NBR1, we performed peptide binding arrays on >100 established LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Comparative analysis of phosphomimetic peptides highlights that while FIP200 and Atg8-family binding are generally augmented by phosphorylation, TAX1BP1 binding is nonresponsive, suggesting differential regulation of these binding events. In vivo studies confirm that LIR-mediated interactions with TAX1BP1 enhance NBR1 activity, increasing autophagosomal delivery by leveraging an additional LIR from TAX1BP1. In sum, these results reveal a one-to-many binding modality in NBR1, providing key insights into the cooperative mechanisms among autophagy receptors. Furthermore, these findings underscore the pervasive role of multifunctional SLiMs in autophagy, offering substantial avenues for further exploration into their regulatory functions.

PKCµ promotes keratinocyte cell migration through Cx43 phosphorylation-mediated suppression of intercellular communication.

Downregulation of intercellular communication through suppression of gap junctional conductance is necessary during wound healing. Connexin 43 (Cx43), a prominent gap junction protein in skin, is downregulated following wounding to restrict communication between keratinocytes. Previous studies found that PKCµ, a novel PKC isozyme, regulates efficient cutaneous wound healing. However, the molecular mechanism by which PKCµ regulates wound healing remains unknown. We have identified that PKCµ suppresses intercellular communication and enhances cell migration in an in vitro wound healing model by regulating Cx43 containing gap junctions. PKCµ can directly interact with and phosphorylate Cx43 at S368, which leads to Cx43 internalization and downregulation. Finally, utilizing phosphomimetic and non-phosphorylatable S368 substitutions and gap junction inhibitors, we confirmed that PKCµ regulates intercellular communication and in vitro wound healing by controlling Cx43-S368 phosphorylation. These results define PKCµ as a critical regulator of Cx43 phosphorylation to control cell migration and wound healing in keratinocytes.

E3 ubiquitin ligases in cancer stem cells: key regulators of cancer hallmarks and novel therapeutic opportunities.

BACKGROUND: Human malignancies are composed of heterogeneous subpopulations of cancer cells with phenotypic and functional diversity. Among them, a unique subset of cancer stem cells (CSCs) has both the capacity for self-renewal and the potential to differentiate and contribute to multiple tumor properties. As such, CSCs are promising cellular targets for effective cancer therapy. At the molecular level, hyper-activation of multiple stemness regulatory signaling pathways and downstream transcription factors play critical roles in controlling CSCs establishment and maintenance. To regulate CSC properties, these stemness pathways are controlled by post-translational modifications including, but not limited to phosphorylation, acetylation, methylation, and ubiquitination. CONCLUSION: In this review, we focus on E3 ubiquitin ligases and their roles and mechanisms in regulating essential hallmarks of CSCs, such as self-renewal, invasion and metastasis, metabolic reprogramming, immune evasion, and therapeutic resistance. Moreover, we discuss emerging therapeutic approaches to eliminate CSCs through targeting E3 ubiquitin ligases by chemical inhibitors and proteolysis-targeting chimera (PROTACs) which are currently under development at the discovery, preclinical, and clinical stages. Several outstanding issues such as roles for E3 ubiquitin ligases in heterogeneity and phenotypical/functional evolution of CSCs remain to be studied under pathologically and clinically relevant conditions. With the rapid application of functional genomic and proteomic approaches at single cell, spatiotemporal, and even single molecule levels, we anticipate that more specific and precise functions of E3 ubiquitin ligases will be delineated in dictating CSC properties. Rational design and proper translation of these mechanistic understandings may lead to novel therapeutic modalities for cancer procession medicine.

Sirtuins: a conserved key unlocking AceCS activity.

Bacterial acetyl-coenzyme A (acetyl-CoA) synthetase (AceCS), an evolutionarily conserved enzyme that converts acetate to acetyl-CoA, is activated by sirtuin-mediated deacetylation. Two recent studies show that this mechanism of regulation is also crucial for mammalian AceCS activity, indicating that control of metabolism at the step of converting acetate to acetyl-CoA is conserved. These findings highlight a metabolic regulatory network controlled by sirtuins that has implications for the mechanisms of calorie restriction and modulation of mammalian lifespan.

Role of Connexin 43 phosphorylation on Serine-368 by PKC in cardiac function and disease.

Intercellular communication mediated by gap junction channels and hemichannels composed of Connexin 43 (Cx43) is vital for the propagation of electrical impulses through cardiomyocytes. The carboxyl terminal tail of Cx43 undergoes various post-translational modifications including phosphorylation of its Serine-368 (S368) residue. Protein Kinase C isozymes directly phosphorylate S368 to alter Cx43 function and stability through inducing conformational changes affecting channel permeability or promoting internalization and degradation to reduce intercellular communication between cardiomyocytes. Recent studies have implicated this PKC/Cx43-pS368 circuit in several cardiac-associated diseases. In this review, we describe the molecular and cellular basis of PKC-mediated Cx43 phosphorylation and discuss the implications of Cx43 S368 phosphorylation in the context of various cardiac diseases, such as cardiomyopathy, as well as the therapeutic potential of targeting this pathway.

Physiological relevance of post-translational regulation of the spindle assembly checkpoint protein BubR1.

BubR1 is an essential component of the spindle assembly checkpoint (SAC) during mitosis where it functions to prevent anaphase onset to ensure proper chromosome alignment and kinetochore-microtubule attachment. Loss or mutation of BubR1 results in aneuploidy that precedes various potential pathologies, including cancer and mosaic variegated aneuploidy (MVA). BubR1 is also progressively downregulated with age and has been shown to be directly involved in the aging process through suppression of cellular senescence. Post-translational modifications, including but not limited to phosphorylation, acetylation, and ubiquitination, play a critical role in the temporal and spatial regulation of BubR1 function. In this review, we discuss the currently characterized post-translational modifications to BubR1, the enzymes involved, and the biological consequences to BubR1 functionality and implications in diseases associated with BubR1. Understanding the molecular mechanisms promoting these modifications and their roles in regulating BubR1 is important for our current understanding and future studies of BubR1 in maintaining genomic integrity as well as in aging and cancer.

Interplay between protein acetylation and ubiquitination controls MCL1 protein stability.

The anti-apoptotic myeloid cell leukemia 1 (MCL1) protein belongs to the pro-survival BCL2 family and is frequently amplified or elevated in human cancers. MCL1 is highly unstable, with its stability being regulated by phosphorylation and ubiquitination. Here, we identify acetylation as another critical post-translational modification regulating MCL1 protein stability. We demonstrate that the lysine acetyltransferase p300 targets MCL1 at K40 for acetylation, which is counteracted by the deacetylase sirtuin 3 (SIRT3). Mechanistically, acetylation enhances MCL1 interaction with USP9X, resulting in deubiquitination and subsequent MCL1 stabilization. Therefore, ectopic expression of acetylation-mimetic MCL1 promotes apoptosis evasion of cancer cells, enhances colony formation potential, and facilitates xenografted tumor progression. We further demonstrate that elevated MCL1 acetylation sensitizes multiple cancer cells to pharmacological inhibition of USP9X. These findings reveal that acetylation of MCL1 is a critical post-translational modification enhancing its oncogenic function and provide a rationale for developing innovative therapeutic strategies for MCL1-dependent tumors.

Genetic fusions favor tumorigenesis through degron loss in oncogenes.

Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis.

Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy.

Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.

The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.

The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic alpha-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.