ATP Analog Research Articles

Structure-Guided Design of Halofuginone Derivatives as ATP-Aided Inhibitors Against Bacterial Prolyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are promising antimicrobial targets due to their essential roles in protein translation, and expanding their inhibitory mechanisms will provide new opportunities for drug discovery. We report here that halofuginone (HF), an herb-derived medicine, moderately inhibits prolyl-tRNA synthetases (ProRSs) from various pathogenic bacteria. A cocrystal structure of Staphylococcus aureus ProRS (SaProRS) with HF and an ATP analog was determined, which guided the design of new HF analogs. Compound 3 potently inhibited SaProRS at IC50 = 0.18 μM and Kd = 30.3 nM and showed antibacterial activities with an MIC of 1-4 μg/mL in vitro. The bacterial drug resistance to 3 only developed at a rate similar to or slower than those of clinically used antibiotics in vitro. Our study indicates that the scaffold and ATP-aided inhibitory mechanism of HF could apply to bacterial ProRS and also provides a chemical validation for using bacterial ProRS as an antibacterial target.

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

Plasmodium species cause malaria and kill hundreds of thousands annually. The microtubule-based motor kinesin-8B is required for development of the flagellated Plasmodium male gamete, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B’s essential role, we characterised the in vitro properties of kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined these motors’ microtubule-bound structures using cryo-electron microscopy, which showed very similar modes of microtubule interaction in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is required for motility and depolymerisation activities of these motors. These data suggest that the mechanochemistry of Plasmodium kinesin-8Bs is functionally tuned to support flagella formation.

Detection of Remdesivir by Liquid Chromatography with Tandem Mass Spectrometry in a Patient with SARS-CoV-2 Infection

One of the officially approved medications for the treatment of the pandemic disease COVID-19, caused by the SARS-CoV-2 virus, is remdesivir. This antiviral molecule is a prodrug that is metabolized into its active form (an ATP analogue). Because of its hepatotoxicity and nephrotoxicity, it is necessary to monitor the serum concentrations of remdesivir. For the therapeutic drug monitoring of remdesivir, a method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization mode was developed. Mass detection was done via triple quadrupole in the Multiple reaction monitoring mode. Separation was done on Zorbax C18 column at 35 °C in mobile phase gradient and flow 0.4 mL min–1 (A – 0.1% formic acid in water, B – 0.1% formic acid in 95% acetonitrile). Time of analysis was 4 minutes. LC-MS/MS method was successfully validated. Calibration was done in blood serum and plasma and it was linear in the range of tested concentrations (0–1000 ng mL–1). Samples were prepared by protein precipitation. The method was used to measure remdesivir concentration in a patient with SARS-CoV-2 infection. The measured concentration 60 minutes after remdesivir application was 175±15 ng mL–1.

Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses.

The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. One major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication, and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, a well-established substrate of pUL97 was no longer hyperphosphorylated. This effect was detectable as early as 4 h post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.

Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Synthesis of the biologically important dideuterium-labelled adenosine triphosphate analogue ApppI(d 2).

The chemical synthesis of the dideuterium-labelled ATP analogue 1-adenosin-5’-yl-3-(3-methylbut-3-en-1,1-d2-1-ol) triphosphoric acid diester (ApppI(d2)) is described. ApppI has been reported to be an important mevalonate pathway metabolite, induced by nitrogen-containing bisphosphonates used for the treatment of several diseases related to the calcium metabolism, of which osteoporosis is the most well-known. The availability of ApppI(d2) opens possibilities to quantitative measurements of ApppI in biological samples by mass spectrometry. The synthesized target compound ApppI(d2) was purified by high-performance counter current chromatography and characterized by 1H, 13C, and 31P NMR spectroscopy as well as high-resolution mass spectrometry.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection via the purinergic P2Y13 receptor and nitric oxide signalling

Abstract Background Red blood cells (RBC) are suggested to act as important mediators in the regulation of cardiovascular function by exporting nitric oxide (NO) bioactivity and ATP under hypoxic/ischemic conditions. In addition, RBCs are known to protect from ischemia-reperfusion injury via the export of NO bioactivity in experimental settings. However, it remains unknown if such beneficial effects of RBCs are protective in patients with acute myocardial infarction. Purpose To investigate whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves activation of purinergic and NO signalling in the RBCs. Methods RBCs were collected from patients with STEMI undergoing primary percutaneous coronary intervention and age- and gender-matched healthy controls. The RBCs were administered into the coronary circulation of isolated Langendorff-perfused rat hearts at the onset of global ischemia for 25 min followed by reperfusion of 60 min. Recovery of left ventricular developed pressure (LVDP) during reperfusion and infarct size were determined. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results Administration of RBCs from STEMI patients improved recovery of LVDP and reduced infarct size in hearts subjected to ischemia-reperfusion in comparison with RBCs from healthy controls (Figure 1A, B). Pre-incubation of the RBCs with the NO synthase (NOS) inhibitor L-NAME (Figure 1C, D) and the inhibitor of the NO receptor soluble guanylyl cyclase (sGC) ODQ abolished the cardioprotective effect of RBCs from STEMI patients. The cardioprotective effect was also attenuated by inhibition of cardiac cGMP-dependent protein kinase (PKG). Further, the purinergic P2Y13 receptor antagonist MRS2211 (Figure 1E, F), but not the P1 receptor antagonist 8PT applied to RBCs, attenuated the cardioprotection induced by RBCs from STEMI patients. Moreover, administration of RBCs from healthy subjects pre-incubated with a cell permeable ATP analogue improved post-ischemic recovery of LVDP and reduced infarct size. This cardioprotective effect was abolished by co-incubation of the RBCs with ODQ (Figure 2) and MRS2211. Conclusion Our findings demonstrate a novel function of RBCs in patients with STEMI that provides protection against myocardial ischemia-reperfusion injury via the activation of P2Y13 receptor and the NO-sGC pathway in RBCs and cardiac PKG. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation; Swedish Research Council

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia–reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia–reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia–reperfusion injury through the P2Y13 receptor and the NO–sGC–PKG pathway.

Association of adenosine signaling gene signature with estrogen receptor-positive breast and prostate cancer bone metastasis.

Bone metastasis is a common and devastating consequence of several major cancer types, including breast and prostate. Osteocytes are the predominant bone cell, and through connexin (Cx) 43 hemichannels release ATP to the bone microenvironment that can be hydrolyzed to adenosine. Here, we investigated how genes related to ATP paracrine signaling are involved in two common bone-metastasizing malignancies, estrogen receptor positive (ER+) breast and prostate cancers. Compared to other sites, bone metastases of both cancer types expressed higher levels of ENTPD1 and NT5E, which encode CD39 and CD73, respectively, and hydrolyze ATP to adenosine. ADORA3, encoding the adenosine A3 receptor, had a similar expression pattern. In primary ER+ breast cancer, high levels of the triplet ENTPD1/NT5E/ADORA3 expression signature was correlated with lower overall, distant metastasis-free, and progression-free survival. In ER+ bone metastasis biopsies, this expression signature is associated with lower survival. This expression signature was also higher in bone-metastasizing primary prostate cancers than in those that caused other tumor events or did not lead to progressive disease. In 3D culture, a non-hydrolyzable ATP analog inhibited the growth of breast and prostate cancer cell lines more than ATP did. A3 inhibition also reduced spheroid growth. Large-scale screens by the Drug Repurposing Hub found ER+ breast cancer cell lines were uniquely sensitive to adenosine receptor antagonists. Together, these data suggest a vital role for extracellular ATP degradation and adenosine receptor signaling in cancer bone metastasis, and this study provides potential diagnostic means for bone metastasis and specific targets for treatment and prevention.

Kinase-Catalyzed Crosslinking and Immunoprecipitation (K-CLIP) to Explore Kinase-Substrate Pairs.

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).

Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

Structural and functional studies of Arabidopsis thaliana triphosphate tunnel metalloenzymes reveal roles for additional domains

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, and inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. In addition, we reveal from crystal structures of AtTTM1 in the presence and absence of a nonhydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates that a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.

Non-Nitrogen-Containing Bisphosphonates Prevent Pyrophosphorylation of Exocytosis Proteins.

Clodronate, a non-nitrogen-containing bisphosphonate (non-NBP), is intracellularly converted into non-hydrolyzable ATP analogs. Clodronate and its analogs impair normal cell functions, including the exocytosis process. However, how this occurs in mast cells is still not well characterized. To summarize the possible mechanisms of clodronate-mediated exocytosis inhibition in mast cells. Non-NBPs display several possible mechanisms of exocytosis inhibition in various cell types, including vesicular nucleotide transporter (VNUT) and purinergic receptor inhibition. Inhibition of purinergic receptors has been shown in mast cells, but VNUT inhibition remains to be confirmed. Inhibition of protein prenylation by non-NBPs has also been shown; however, direct evidence of non-NBPs in prenylated exocytosis proteins is still contradictory. Finally, non-NBPs may inhibit mast cell exocytosis via impairment of protein pyrophosphorylation. This mechanism is less studied, and direct evidence of the involvement of pyrophosphorylated proteins in exocytosis is still lacking. Non-NBPs may affect mast cell exocytosis by interacting with purinergic receptors or VNUT or by preventing post-translational modifications of exocytosis protein(s), i.e., prenylation and pyrophosphorylation. The latter needs further investigation to provide direct evidence of a role for non- NBPs.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more ‘open’ allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P–31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

Structural basis for heme detoxification by an ATP-binding cassette–type efflux pump in gram-positive pathogenic bacteria

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.

The 26S Proteasome Switches between ATP-Dependent and -Independent Mechanisms in Response to Substrate Ubiquitination.

The ubiquitin–proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.

P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7-/- CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

P2RX7 enhances tumor control by CD8+ T cells in adoptive cell therapy

Abstract Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes generation of memory populations following acute infections and is critical for responses to chronic infection. Despite these findings, recent studies have indicated that P2RX7 may limit the efficacy of anti-tumor responses. We show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T cell adoptive transfer model. Tumor specific P2rx7−/− CD8+ T cells exhibited impaired mitochondrial maintenance and function, but do not display signs of overt exhaustion or poor survival early in the anti-tumor response. However, as tumor burden increases the frequency P2RX7 deficient CD8+ T cells progressively declines relative to WT CD8+ T cells within the tumor. This was associated with worsening mitochondrial function and increased expression of some markers of T cell exhaustion among P2rx7−/− CD8+ T cells. Extending these studies, we found that transient in vitro stimulation of P2RX7 using the ATP analog BzATP led to enhanced B16 tumor control by CD8+ T cells. The improved ability of BzATP stimulated CD8+ T cells to prolong survival of tumor bearing mice was accompanied by enhanced mitochondrial function of tumor specific CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and indicates the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy. Supported by grants F30 award from the NIH (1F30CA250321-01)

The 26S Proteasome Switches Between ATP‐Dependent and Independent Mechanisms in Response to Substrate Ubiquitination

The Ubiquitin‐Proteasome System is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome by the attachment of polyubiquitin chains. A number of proteins also contain ubiquitin‐independent degrons (UbID) that allow for proteasomal targeting without the need for ubiquitination. UbID substrates are degraded less processively than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust with complete degradation only occurring with relatively unstable substrates (those that were at least transiently unfolded as determined by protease sensitivity). Surprisingly, UbID degradation was unaffected by the non‐hydrolyzable ATP analog ATPγS, which halts ATP‐dependent engagement and translocation of substrates. Furthermore, ubiquitin‐dependent degradation proceeded in a strikingly similar fashion to UbID degradation in the presence of ATPγS, suggesting the 26S proteasome may have a “default” ATP‐ and ubiquitin‐independent mechanism that is capable of unfolding and degrading less‐stable proteins.