FOXP3-expressing regulatory T （Treg） cells, which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. Infiltration of a large number of Treg cells into tumor tissues is often associ- ated with poor prognosis. There is accumulating evidence that the removal of Treg cells is able to evoke and enhance anti-tumor immune response. However, systemic depletion of Treg cells may concurrently elicit deleterious autoim- munity. One strategy for evoking effective tumor immunity without antoimmunity is to specifically target terminally differentiated effector Treg cells rather than all FOXP3＋ T cells, because effector Treg cells are the predominant cell type in tumor tissues. Various cell surface molecules, including chemokine receptors such as CCR4, that are specifi- cally expressed by effector Treg cells can be the candidates for depleting effector Treg cells by specific cell-depleting monoclonal antibodies. In addition, other immunological characteristics of effector Treg cells, such as their high ex- pression of CTLA-4, active proliferation, and apoptosis-prone tendency, can be exploited to control specifically their functions. For example, anti-CTLA-4 antibody may kill effector Treg ceils or attenuate their suppressive activity. It is hoped that combination of Treg-cell targeting （e.g., by reducing Treg cells or attenuating their suppressive activity in tumor tissues） with the activation of tumor-specific effector T cells （e.g., by cancer vaccine or immune checkpoint blockade） will make the current cancer immunotherapy more effective.
Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modi- fications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the ＇ubiquitin code＇. Ubiquitin can be ubiquitinated on seven lysine （Lys） residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiq- uitin-like molecules （such as SUMO or NEDD8）. Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiqnitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.
The recent Zika virus （ZIKV） epidemic in Latin America coincided with a marked increase in microcephaly in newborns. However, the causal link between maternal ZIKV infection and malformation of the fetal brain has not been firmly established. Here we show a vertical transmission of ZIKV in mice and a marked effect on fetal brain development. We found that intraperitoneal （i.p.） injection of a contemporary ZIKV strain in pregnant mice led to the infection of radial gila cells （RGs） of dorsal ventricular zone of the fetuses, the primary neural progenitors responsi- ble for cortex development, and caused a marked reduction of these cortex founder cells in the fetuses. Interestingly, the infected fetal mice exhibited a reduced cavity of lateral ventricles and a discernable decrease in surface areas of the cortex. This study thus supports l;he conclusion that vertically transmitted ZIKV affects fetal brain development and provides a valuable animal model for the evaluation of potential therapeutic or preventative strategies.
Single-ceU genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple om- ics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations （CNVs）, DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs gen- erally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.
Ferroptosis is an iron-dependent form of regulated necrosis. It is implicated in various human diseases, including ischemic organ damage and cancer. Here, we report the crucial role of autophagy, particularly autophagic degradation of cellular iron storage proteins （a process known as ferritinophagy）, in ferroptosis. Using RNAi screening coupled with subsequent genetic analysis, we identified multiple autophagy-related genes as positive regulators of ferroptosis. Ferroptosis induction led to autophagy activation and consequent degradation of ferritin and ferritino- phagy cargo receptor NCOA4. Consistently, inhibition of ferritinophagy by blockage of autophagy or knockdown of NCOA4 abrogated the accumulation of ferroptosis-associated cellular labile iron and reactive oxygen species, as well as eventual ferroptotic cell death. Therefore, ferroptosis is an autophagic cell death process, and NCOA4-mediated ferritinophagy supports ferroptosis by controlling cellular iron homeostasis.
Dynamic modulation of protein levels is tightly controlled in response to physiological cues. In mammalian cells, much of the protein degradation is carried out by the ubiquitin-proteasome system （UPS）. Similar to kinases, com- ponents of the ubiquitin system are often dysregulated, leading to a variety of diseases, including cancer and neuro- degeneration, making them attractive drug targets. However, so far there are only a handful of drugs targeting the ubiquitin system that have been approved by the FDA. Here, we review possible therapeutic intervention nodes in the ubiquitin system, analyze the challenges, and highlight the most promising strategoies to targoet the UPS.
Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions （termed pyroptotic bodies） prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like （MLKL） oligomers in the plasma membrane, whereas gasdermin-D （GSDMD） mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD （GSDMD-N） generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.
Chimeric antigen receptor （CAR） gene-engineered T cell therapy holds the potential to make a meaningful differ- ence in the lives of patients with terminal cancers. For decades, cancer therapy was based on biophysical parameters, with surgical resection to debulk, followed by radiation and chemotherapy to target the rapidly growing tumor cells, while mostly sparing quiescent normal tissues. One breakthrough occurred with allogeneic bone-marrow transplant for patients with leukemia, which provided a sometimes curative therapy. The field of adoptive cell therapy for sol- id tumors was established with the discovery that tumor-infiltrating lymphocytes could be expanded and used to treat and even cure patients with metastatic melanoma. Tumor-specific T-cell receptors （TCRs） were identified and engineered into patient peripheral blood lymphocytes, which were also found to treat tumors. However, these were limited by patient HLA-restriction. Close behind came generation of CAR, combining the exquisite recognition of an antibody with the effector function of a T cell. The advent of CD19-targeted CARs for treating patients with multiple forms of advanced B-cell malignancies met with great success, with up to 95% response rates. Applying CAR treat- ment to solid tumors, however, has just begun, but already certain factors have been made clear： the tumor target is of utmost importance for clinicians to do no harm; and solid tumors respond differently to CAR therapy compared with hematologic ones. Here we review the state of clinical gene-engineered T cell immunotherapy, its successes, chal- lenges, and future.
Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion （DRG） by high-coverage single-cell RNA-sequencing （10 950 ±1 218 genes per neuron） and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors （MHNs）. Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors （MNs） expressing BAil-associated protein 2-like I （Baiap211）. Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap211-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.
Immunotherapy using dendritic cell （DC）-based vaccination is an approved approach for harnessing the potential of a patient＇s own immune system to eliminate tumor cells in metastatic hormone-refractory cancer. Overall, al- though many DC vaccines have been tested in the clinic and proven to be immunogenic, and in some cases associated with clinical outcome, there remains no consensus on how to manufacture DC vaccines. In this review we will discuss what has been learned thus far about human DC biology from clinical studies, and how current approaches to apply DC vaccines in the clinic could be improved to enhance anti-tumor immunity.
The origin and evolution of the domestic dog remains a controversial question for the scientific community, with basic aspects such as the place and date of origin, and the number of times dogs were domesticated, open to dispute. Using whole genome sequences from a total of 58 canids （12 gray wolves, 27 primitive dogs from Asia and Africa, and a collection of 19 diverse breeds from across the world）, we find that dogs from southern East Asia have significantly higher genetic diversity compared to other populations, and are the most basal group relating to gray wolves, indicating an ancient origin of domestic dogs in southern East Asia 33 000 years ago. Around 15 000 years ago, a subset of ancestral dogs started migrating to the Middle East, Africa and Europe, arriving in Europe at about 10 000 years ago. One of the out of Asia lineages also migrated back to the east, creating a series of admixed populations with the endemic Asian lineages in northern China before migrating to the New World. For the first time, our study unravels an extraordinary journey that the domestic dog has traveled on earth.
Ubiquitin-conjugating enzymes （E2s） are the central players in the trio of enzymes responsible for the attachment of ubiquitin （Ub） to cellular proteins. Humans have -40 E2s that are involved in the transfer of Ub or Ub-like （Ubl） proteins （e.g., SUMO and NEDDS）. Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s.
SIRT6 belongs to the mammalian homologs of Sir2 histone NAD＋-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout hu- man mesenchymal stem cells （hMSCs） by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated func- tional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sen- sitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 （NRF2） and RNA polymerase II, which was required for the transactivation of NRF2-regulated an- tioxidant genes, including heme oxygenase 1 （HO-1）. Overexpression of HO-1 in SIRT6-null hMSCs rescued prema- ture cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.
A pre-existing T cell-inflamed tumor microenvironment has prognostic utility and also can be predictive for re- sponse to contemporary cancer immunotherapies. The generation of a spontaneous T cell response against tumor-as- sociated antigens depends on innate immune activation, which drives type I interferon （IFN） production. Recent work has revealed a major role for the STING pathway of cytosolic DNA sensing in this process. This cascade of events contributes to the activation of Batf3-1ineage dendritic cells （DCs）, which appear to be central to anti-tumor immunity. Non-T cell-inflamed tumors lack chemokines for Batf3 DC recruitment, have few Batf3 DCs, and lack a type I IFN gene signature, suggesting that failed innate immune activation may be the ultimate cause for lack of spontaneous T cell activation and accumulation. With this information in hand, new strategies for triggering innate immune activation and Batf3 DC recruitment are being developed, including novel STING agonists for de novo im- mune priming. Ultimately, the successful development of effective innate immune activators should expand the frac- tion of patients that can respond to immunotherapies, such as with checkpoint blockade antibodies.
MicroRNAs （miRNAs） are critical regulators of gene expression, and exert extensive impacts on development, physi- ology, and disease of eukaryotes. A high degree of parallelism is found in the molecular basis of miRNA biogenesis and action in plants and animals. Recent studies interestingly suggest a potential cross-kingdom action of plant-de- rived miRNAs, through dietary intake, in regulating mammalian gene expression. Although the source and scope of plant miRNAs detected in mammalian specimens remain controversial, these initial studies inspired us to determine whether plant miRNAs can be detected in Western human sera and whether these plant miRNAs are able to influ- ence gene expression and cellular processes related to human diseases such as cancer. Here we found that Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients. In human sera, miR159 was predominantly detected in the extracellular vesicles, and was resistant to sodium periodate oxidation suggesting the plant-originated 2＇-O-methylation on the 3＇ terminal ribose. In breast cancer cells but not non-cancerous mammary epithelial cells, a synthetic mimic of miR159 was capable of inhibiting proliferation by targeting TCF7 that encodes a Wnt signaling transcription factor, leading to a decrease in MYC protein levels. Oral administration of miR159 mimic significantly suppressed the growth of xenograft breast tumors in mice. These results demonstrate for the first time that a plant miRNA can inhibit cancer growth in mammals.
Ubiquitination has emerged as a crucial mechanism that regulates signal transduction in diverse biological pro- cesses, including different aspects of immune functions. Ubiquitination regulates pattern-recognition receptor sig- naling that mediates both innate immune responses and dendritic cell maturation required for initiation of adaptive immune responses. Ubiquitination also regulates the development, activation, and differentiation of T cells, thereby maintaining efficient adaptive immune responses to pathogens and immunological tolerance to self-tissues. Like phosphorylation, ubiquitination is a reversible reaction tightly controlled by the opposing actions of ubiquitin ligases and deubiquitinases. Deregulated ubiquitination events are associated with immunological disorders, including auto- immune and inflammatory diseases.
The evolutionarily conserved target of rapamycin complex 1 （TORC1） is a master regulator of cell growth and metabolism. In mammals, growth factors and cellular energy stimulate mTORC1 activity through inhibition of the TSC complex （TSC1-TSC2-TBC1D7）, a negative regulator of mTORC1. Amino acids signal to mTORC1 independently of the TSC complex. Here, we review recently identified regulators that link amino acid sufficiency to mTORC1 activity and how mutations affecting these regulators cause human disease.
Harnessing the immune system to eradicate malignant cells is becoming a most powerful new approach to cancer therapy. FDA approval of the immunotherapy-based drugs, sipuleucel-T （Provenge）, ipilimumab （Yervoy, anti-CT- LA-4）, and more recently, the programmed cell death （PD）-I antibody （pembroliznmab, Keytruda）, for the treatment of multiple types of cancer has greatly advanced research and clinical studies in the field of cancer immunotherapy. Furthermore, recent clinical trials, using NY-ESO-l-specific T cell receptor （TCR） or CD19-chimeric antigen re- ceptor （CAR）, have shown promising clinical results for patients with metastatic cancer. Current success of cancer immunotherapy is built upon the work of cancer antigens and co-inhibitory signaling molecules identified 20 years ago. Among the large numbers of target antigens, CD19 is the best target for CAR T cell therapy for blood cancer, but CAR-engineered T cell immunotherapy does not yet work in solid cancer. NY-ESO-1 is one of the best targets for TCR-based immunotherapy in solid cancer. Despite the great success of checkpoint blockade therapy, more than 50% of cancer patients fail to respond to blockade therapy. The advent of new technologies such as next-generation sequencing has enhanced our ability to search for new immune targets in onco-immunology and accelerated the de- velopment of immunotherapy with potentially broader coverage of cancer patients. In this review, we will discuss the recent progresses of cancer immunotherapy and novel strategies in the identification of new immune targets and mu- tation-derived antigens （neoantigens） for cancer immunotherapy and immunoprecision medicine.
The 26S proteasome is a large, -2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms.
CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRIS-PR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.