Johannes Elferich

Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context.

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT–substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins

Significance ROS1 fusion kinases are critical oncogenes in several malignancies, suggesting that ROS1 inhibitors are likely to be effective molecularly targeted therapies in these patients. Although phase I/II clinical trials using the ALK/ROS1 inhibitor crizotinib to treat ROS1 fusion-harboring non–small-cell lung cancer patients demonstrate early success, evidence of clinical resistance to crizotinib due to the acquired ROS1G2032R mutation was recently reported. Here, we demonstrate that foretinib is a more potent ROS1 inhibitor than crizotinib in vitro and in vivo and remains effective against crizotinib-resistant ROS1 kinase domain mutations, including ROS1 G2032R. Taken together, our findings establish foretinib as a highly promising therapeutic candidate for treating patients with ROS1-driven malignancies and provide rationale for rapid clinical translation. The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non–small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1G2032R mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.

Monoubiquitination Is Critical for Ovarian Tumor Domain-containing Ubiquitin Aldehyde Binding Protein 1 (Otub1) to Suppress UbcH5 Enzyme and Stabilize p53 Protein

Background: Otub1 suppresses E2 UbcH5 to stabilize and activate p53. Results: UbcH5 monoubiquitinates Otub1, and monoubiquitination-defective Otub1 mutants fail to inhibit UbcH5 and induce p53. Conclusion: Monoubiquitination is critical for Otub1 to suppress UbcH5 and induce p53. Significance: We report the discovery of a novel molecular mechanism underlying Otub1 suppression of E2 and activation of p53. Ovarian tumor domain-containing ubiquitin (Ub) aldehyde binding protein 1 (Otub1) regulates p53 stability and activity via non-canonical inhibition of the MDM2 cognate Ub-conjugating enzyme (E2) UbcH5. However, it is not clear how this activity of Otub1 is regulated in cells. Here we report that Otub1 is monoubiquitinated by UbcH5 in cells and in vitro, primarily at the lysine 59 and 109 residues. This monoubiquitination, in turn, contributes to the activity of Otub1 to suppress UbcH5. The lysine-free Otub1 mutant (Otub1K0) fails to be monoubiquitinated and is unable to suppress the Ub-conjugating activity of UbcH5 in vitro and the MDM2-mediated p53 ubiquitination in cells. Consistently, this mutant is unable to stabilize p53, induce apoptosis, and suppress cell proliferation. Overexpression of Otub1K0 inhibits DNA-damage induced apoptosis. Adding either Lys-59 or Lys-109 back to the Otub1K0 mutant restores the monoubiquitination of Otub1 and its function to stabilize and activate p53. We further show that UbcH5 preferentially binds to the monoubiquitinated Otub1 via Ub interaction with its backside donor Ub-interacting surface, suggesting that this binding interferes with the self-assembly of Ub-charged UbcH5 (UbcH5∼Ub) conjugates, which is critical for Ub transfer. Thus, our data reveal novel insights into the Otub1 inhibition of E2 wherein monoubiquitination promotes the interaction of Otub1 with UbcH5 and the function to suppress it.

Cotranslational folding inhibits translocation from within the ribosome–Sec61 translocon complex

Eukaryotic secretory proteins cross the endoplasmic reticulum (ER) membrane through a protein-conducting channel contained within the ribosome–Sec61translocon complex (RTC). Using a zinc-finger sequence as a folding switch, we show that cotranslational folding of a secretory passenger inhibits translocation in canine ER microsomes and in human cells. Folding occurs within a cytosolically inaccessible environment, after ER targeting but before initiation of translocation, and it is most effective when the folded domain is 15–54 residues beyond the signal sequence. Under these conditions, substrate is diverted into cytosol at the stage of synthesis in which unfolded substrate enters the ER lumen. Moreover, the translocation block is reversed by passenger unfolding even after cytosol emergence. These studies identify an enclosed compartment within the assembled RTC that allows a short span of nascent chain to reversibly abort translocation in a substrate-specific manner.

Identification of the Intracellular Gate for a Member of the Equilibrative Nucleoside Transporter (ENT) Family

Background: Equilibrative nucleoside transporters (ENTs) play important roles in biology and disease. Results: The intracellular gate for an ENT, LdNT1.1, has been identified by computational and experimental methods. Conclusion: The intracellular gate consists of the ends of four transmembrane helices. Significance: An important functional component of ENTs has been identified. Equilibrative nucleoside transporters of the SLC29 family play important roles in many physiological and pharmacological processes, including import of drugs for treatment of cancer, AIDS, cardiovascular, and parasitic diseases. However, no crystal structure is available for any member of this family. In previous studies we generated a computational model of the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1) that captured this permease in the outward-closed conformation, and we identified the extracellular gate. In the present study we have modeled the inward-closed conformation of LdNT1.1 using the crystal structure of the Escherichia coli fucose transporter FucP and have identified four transmembrane helices whose ends close to form a predicted intracellular gate. We have tested this prediction by site-directed mutagenesis of relevant helix residues and by cross-linking of introduced cysteine pairs. The results are consistent with the predictions of the computational model and suggest that a similarly constituted gate operates in other members of the equilibrative nucleoside transporter family.

Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

Background: Histidine 69 in the propeptide is a pH sensor that mediates compartment-specific furin activation. Results: Histidine 69 protonation exposes the activation loop for proteolysis only within an optimal window for pH-dependent activation. Conclusion: A small structural change functions as the trigger that regulates precise spatiotemporal activation of furin. Significance: Our work provides insights into how individual proprotein convertases encode their unique compartment-specific activation. The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70–80% of patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C‐terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium‐binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL‐3‐independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2‐V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll‐like receptor agonist. These effects are not dependent on the novel C‐terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. Am. J. Hematol. 91:211–219, 2016.

KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

Background: The mechanism for selectively targeting membrane proteins to the flagellum of kinetoplastid parasites is unknown. Results: We have identified a novel protein, KHARON1, which is important for the flagellar targeting of a glucose transporter. Conclusion: KHARON1 is the first protein identified in Kinetoplastida that targets a membrane protein to the flagellum. Significance: KHARON1 may be part of a new flagellar targeting pathway. The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

Substrate inhibition of uracil phosphoribosyltransferase by uracil can account for the uracil growth sensitivity of Leishmania donovani pyrimidine auxotrophs.

Background: Leishmania donovani salvage all pyrimidines through uracil phosphoribosyltransferase (LdUPRT). Results: LdUPRT phosphoribosylates uracil, 5-fluorouracil, and 4-thiouracil and is susceptible to substrate inhibition. Conclusion: LdUPRT recognizes pyrimidine analogs, and substrate inhibition by LdUPRT explains the supersensitivity of pyrimidine auxotrophs to uracil. Significance: Substrate inhibition of LdUPRT provides a mechanism for uracil susceptibility and offers a protective function for the parasite. The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μm for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.