Genetically incorporated crosslinkers identify regulators of membrane protein PD-L1 in mammalian cells.

Profiling membrane proteins' interacting networks is crucial for understanding their regulatory mechanisms and functional characteristics, but it remains a challenging task. Here, by combining genetic incorporation of crosslinkers, tandem denatured purification, and proteomics, we added interaction partners for PD-L1, a cancer cell surface protein that inhibits T cell activity. The site-specifically incorporated crosslinker mediates the covalent capture of interactions under physiological conditions and enabled the PD-L1 complexes to withstand the harsh extraction conditions of membrane proteins. Subsequent experiments led to the identification of potential PD-L1 interaction candidates and verified membrane-associated progesterone receptor component 1 as a novel PD-L1 interaction partner in mammalian cells. Importantly, we demonstrated that PGRMC1 positively regulates PD-L1 expression by regulating GSK3ß-mediated PD-L1 degradation in cancer cells. Furthermore, PGRMC1 knockdown results in dramatically enhanced T cell-mediated cytotoxicity in cancer cells. In conclusion, our study elucidated the interactome of PD-L1 and uncovered a new player in the PD-L1 regulation mechanism.

Bacterial effector restricts liquid-liquid phase separation of ZPR1 to antagonize host UPRER.

How pathogens manipulate host UPRER to mediate immune evasion is largely unknown. Here, we identify the host zinc finger protein ZPR1 as an interacting partner of the enteropathogenic E. coli (EPEC) effector NleE using proximity-enabled protein crosslinking. We show that ZPR1 assembles via liquid-liquid phase separation (LLPS) in vitro and regulates CHOP-mediated UPRER at the transcriptional level. Interestingly, in vitro studies show that the ZPR1 binding ability with K63-ubiquitin chains, which promotes LLPS of ZPR1, is disrupted by NleE. Further analyses indicate that EPEC restricts host UPRER pathways at the transcription level in a NleE-ZPR1 cascade-dependent manner. Together, our study reveals the mechanism by which EPEC interferes with CHOP-UPRER via regulating ZPR1 to help pathogens escape host defense.

Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells.

Whether membrane-anchored PD-L1 homodimerizes in living cells is controversial. The biological significance of the homodimer waits to be expeditiously explored. However, characterization of the membrane-anchored full-length PD-L1 homodimer is challenging, and unconventional approaches are needed. By using genetically incorporated crosslinkers, we showed that full length PD-L1 forms homodimers and tetramers in living cells. Importantly, the homodimerized intracellular domains of PD-L1 play critical roles in its complex glycosylation. Further analysis identified three key arginine residues in the intracellular domain of PD-L1 as the regulating unit. In the PD-L1/PD-L1-3RE homodimer, mutations result in a decrease in the membrane abundance and an increase in the Golgi of wild-type PD-L1. Notably, PD-1 binding to abnormally glycosylated PD-L1 on cancer cells was attenuated, and subsequent T-cell induced toxicity increased. Collectively, our study demonstrated that PD-L1 indeed forms homodimers in cells, and the homodimers play important roles in PD-L1 complex glycosylation and T-cell mediated toxicity.

Genetically incorporated crosslinkers reveal NleE attenuates host autophagy dependent on PSMD10.

Autophagy acts as a pivotal innate immune response against infection. Some virulence effectors subvert the host autophagic machinery to escape the surveillance of autophagy. The mechanism by which pathogens interact with host autophagy remains mostly unclear. However, traditional strategies often have difficulty identifying host proteins that interact with effectors due to the weak, dynamic, and transient nature of these interactions. Here, we found that Enteropathogenic Escherichia coli (EPEC) regulates autophagosome formation in host cells dependent on effector NleE. The 26S Proteasome Regulatory Subunit 10 (PSMD10) was identified as a direct interaction partner of NleE in living cells by employing genetically incorporated crosslinkers. Pairwise chemical crosslinking revealed that NleE interacts with the N-terminus of PSMD10. We demonstrated that PSMD10 homodimerization is necessary for its interaction with ATG7 and promotion of autophagy, but not necessary for PSMD10 interaction with ATG12. Therefore, NleE-mediated PSMD10 in monomeric state attenuates host autophagosome formation. Our study reveals the mechanism through which EPEC attenuates host autophagy activity.

LMR-101, a novel derivative of propofol, exhibits potent anticonvulsant effects and possibly interacts with a novel target on γ-aminobutyric acid type A receptors.

OBJECTIVE: LMR-101 is a bisphenol derivative of propofol, a short-acting general anesthetic, which is also used to manage status epilepticus (SE). We evaluated the sedative and anticonvulsant effects of LMR-101 to discover its potential to manage epilepsy and SE in the clinic. METHODS: Comparative studies between LMR-101 and propofol were performed in mice to elucidate an appropriate dose range for LMR-101 that produced anticonvulsant effects without significant sedation. Then, the anticonvulsive efficacy for LMR-101 was evaluated using seizure models induced by pentylenetetrazol and (+)-bicuculline. The ability of LMR-101 to inhibit SE was assessed using a rat model of SE induced by pilocarpine. Radioligand binding assay profiles for LMR-101 were performed to evaluate the potential mechanisms of action underlying its anticonvulsant properties. RESULTS: In the mouse study, LMR-101 exhibited greater anticonvulsant and lesser sedative effect compared with propofol. LMR-101 completely inhibited pentylenetetrazol-induced seizures at a dose of 50 mg/kg and exhibited heavy sedation at 300 mg/kg. Propofol anesthetized all mice and only decreased the seizure rate at 25 mg/kg. LMR-101 also suppressed seizure behaviors evoked by (+)-bicuculline in mice in a dose-dependent manner. In the pilocarpine-induced SE model, LMR-101 significantly decreased the maximum seizure score and seizure duration in a dose-dependent manner. The median effective dose for LMR-101 was 14.30 mg/kg and 121.87 mg/kg to prevent and inhibit sustained SE, respectively. In binding assays, LMR-101 primarily inhibited tert-[35 S] butylbicyclophosphorothionate binding to γ-aminobutyric acid type A (GABAA ) receptors (half-maximal inhibitory concentration = 2.06 µmol·L-1 ), but it did not affect [3 H] flunitrazepam or [3 H] muscimol binding. SIGNIFICANCE: It is anticipated that LMR-101 might play an essential role in the clinical management of epilepsy and SE. LMR-101 also might bind to a novel target site on the GABAA receptor that is different from existing antiepileptic drugs. Further study of the mechanisms of action of LMR-101 would be of considerable value in the search for new active drug sites on GABAA receptors.

Dissociation between Iron and Heme Biosyntheses Is Largely Accountable for Respiration Defects of Shewanella oneidensis fur Mutants.

Iron, a major protein cofactor, is essential for most organisms but can simultaneously be toxic. Iron homeostasis thus has to be effectively maintained under a range of iron regimes. This may be particularly true with Shewanella oneidensis, a representative of dissimilatory metal-reducing bacteria (DMRB), which are capable of respiring a variety of chemicals as electron acceptors (EAs), including iron ores. Although iron respiration and its regulation have been extensively studied in this bacterium, how iron homeostasis is maintained remains largely unknown. Here, we report that the loss of the iron homeostasis master regulator Fur negatively affects the respiration of all EAs tested. This defect appears mainly to be a result of reduced cytochrome c (cyt c) production, despite a decrease in the expression of reductases that are under the direct control of Fur. We also show that S. oneidensis Fur interacts with canonical Fur box motifs in F-F-x-R configuration rather than the palindromic motif proposed before. The fur mutant has lowered total iron and increased free iron contents. Under iron-rich conditions, overproduction of the major iron storage protein Bfr elevates the total iron levels of the fur mutant over those of the wild-type but does not affect free iron levels. Intriguingly, such an operation only marginally improves cyt c production by affecting heme b biosynthesis. It is established that iron dictates heme b/cyt c biosynthesis in S. oneidensisfur+ strains, but the fur mutation annuls the dependence of heme b/cyt c biosynthesis on iron. Overall, our results suggest that Fur has a profound impact on the iron homeostasis of S. oneidensis, through which many physiological processes, especially respiration, are transformed.IMPORTANCE Iron reduction is a signature of S. oneidensis, and this process relies on a large number of type c cytochromes, which per se are iron-containing proteins. Thus, iron plays an essential and special role in iron respiration, but to date, the nature of iron metabolism and regulation of the bacterium remains largely unknown. In this study, we investigated impacts of Fur, the master regulator of iron homeostasis, on respiration. The loss of Fur causes a general defect in respiration, a result of impaired cyt c production rather than specific regulation. Additionally, the fur mutant is unresponsive to iron, resulting in imbalanced iron homeostasis and dissociation between iron and cyt c production. These findings provide important insights into the iron biology of DMRB.

Investigation of a spontaneous mutant reveals novel features of iron uptake in Shewanella oneidensis.

Shewanella oneidensis is among the first and the best studied bacteria capable of respiring minerals as terminal electron acceptors (EAs), including a variety of iron ores. This respiration process relies on a large number of c-type cytochromes, which per se are iron-containing proteins. Thus, iron plays an essential and special role in iron respiration of S. oneidensis, prompting extensive investigations into iron physiology. Despite this, we still know surprisingly little about the components and characteristics of iron transport in this bacterium. Here, we report that TonB-dependent receptor PutA (SO_3033) is specific to the siderophore-mediated iron uptake. Although homologs of PutA are abundant, none of them can function as a replacement. In the absence of PutA, S. oneidensis suffers from an iron shortage, which leads to a severe defect in production of cytochrome c. However, proteins requiring other types of cytochromes, such as b and d, do not appear to be significantly impacted. Intriguingly, lactate, but not other carbon sources that are routinely used to support growth, is able to promote iron uptake when PutA is missing. We further show that the lactate-mediated iron import is independent of lactate permeases. Overall, our results suggest that in S. oneidensis the siderophore-dependent pathway plays a key role in iron uptake when iron is limited, but many alternative routes exist.