extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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TrkAIII signals endoplasmic reticulum stress to the mitochondria in neuroblastoma cells, resulting in glycolytic metabolic adaptation

Alternative TrkAIII splicing characterises advanced stage metastatic disease and post-therapeutic relapse in neuroblastoma (NB), and in NB models TrkAIII exhibits oncogenic activity. In this study, we report a novel role for TrkAIII in signaling ER stress to the mitochondria in SH-SY5Y NB cells that results in glycolytic metabolic adaptation. The ER stress-inducing agents DTT, A23187 and thapsigargin activated the ER stress-response in control pcDNA SH-SY5Y and TrkAIII expressing SH-SY5Y cells and in TrkAIII SH-SY5Y cells increased TrkAIII targeting to mitochondria and internalisation into inner-mitochondrial membranes. Within inner-mitochondrial membranes, TrkAIII was subjected to Omi/HtrA2-dependent cleavage to tyrosine phosphorylated 45–48kDa carboxyl terminal active fragments, localised predominantly in tyrosine kinase-domain mitochondrial matrix orientation. This stress-induced activation of mitochondrial TrkAIII was associated with increased ROS production, prevented by the ROS scavenger Resveratrol and underpinned by changes in Ca2+ movement, implicating ROS/Ca2+ interplay in overcoming the mitochondrial TrkAIII activation threshold. Stress-induced, cleavage-activation of mitochondrial TrkAIII resulted in mitochondrial PDHK1 tyrosine phosphorylation, leading to glycolytic metabolic adaptation. This novel mitochondrial role for TrkAIII provides a potential self-perpetuating, drug reversible way through which tumour microenvironmental stress may maintain the metastasis promoting “Warburg effect” in TrkAIII expressing NBs.

INTRODUCTION
Alternative TrkAIII splicing of the neurotrophin receptor gene TrkA in neuroblastoma (NB) is characterised by exon 6-7 skipping, associates with advanced stage metastatic disease and post-therapeutic relapse, and in NB models TrkAIII exhibits oncogenic activity and promotes chemotherapeutic resistance [1][2][3][4][5][6][7][8]. The TrkAIII oncoprotein is devoid of the D4 activationprevention domain [1,9] and several N-glycosylation sites important for cell surface receptor localisation [1,10]. As a consequence, TrkAIII is not expressed at the cell surface but accumulates within pre-Golgi membranes and at the centrosome, where it exhibits spontaneous ligand-independent activation. Spontaneous intracellular TrkAIII activation leads to chronic signaling through the IP3K/Akt but not RAS/MAPK pathway and promotes a more stem cell-like, anaplastic, pro-angiogenic, stress-resistant, genetically unstable, tumourigenic and metastatic phenotype [1-3, 6, 7, 11-13]. In NB cell lines, alternative TrkAIII splicing is promoted by a hypoxia mimic, suggesting that it represents a mechanism through which tumour suppressing signals from fully spliced TrkA receptors can switch to tumor promoting signals from Research Paper www.impactjournals.com/oncotarget TrkAIII within the hypoxic tumour microenvironment [1,2,6]. Furthermore, spontaneous activation of TrkAIII within the ERGIC-COP1 compartment and at the centrosome provides novel alternatives to "classical" cell surface oncogenic receptor tyrosine kinase (RTK) signaling and fuels the growing hypothesis that the RTK oncoprotein mislocalization underpins oncogenic activity [11,14,15].
Stress within the tumour microenvironment promotes tumour progression by selecting resistant tumour cells that are protected against stress-induced death by conserved physiological stress-protection mechanisms, activated oncogenes and the loss of tumour suppressors. The endoplasmic reticulum stress response (ERSR) represents one such mechanism that is conserved by tumour cells and utilised for adaptation and survival within the stressful tumour microenvironment [16]. The ERSR is activated by the accumulation of damaged, underglycosylated and/or misfolded proteins within the ER and is induced by hypoxia, acidosis and nutrient deprivation, all of which characterise the tumour microenvironment. Damaged, misfolded and/or aggregated proteins accumulating within the ER competitively bind the ER chaperone Grp78/Bip, which dissociates from the ER stress-response factors ATF6, Ire1α and PERK. These factors are subsequently activated and orchestrate an adaptive response that reduces protein translation, increases ER storage capacity, eliminates damaged proteins, re-folds misfolded proteins, alters metabolism and protects against ER stress-induced death [16,17].
The ER also communicates with mitochondria via specialised mitochondrial-associated ER membrane (MAM) sites. These sites regulate the flow of Ca 2+ , proteins and lipids between the ER and mitochondria [18,19]. ER stress causes the release of Ca 2+ from the ER lumen [20] and increases mitochondrial Ca 2+ uptake. Mitochondrial Ca 2+ is critical for respiratory function, optimises respiratory enzyme activity and regulates mitochondrial ROS production [20,21] but elevated levels of mitochondrial Ca 2+ have potential to increase mitochondrial ROS production to damaging levels [20][21][22][23][24][25][26][27]. Under such conditions, the fate of mitochondria is regulated by redox enzyme systems, superoxide dismutases, the inter-membrane space serine protease Omi/HtrA2 [28][29][30][31][32] and also by the mitochondrial unfolded protein response (mt-UPR). The mt-UPR activates an independent transcriptional program that enhances mitochondrial survival through metabolic adaptation, proteolytic elimination of damaged proteins and selective elimination of damaged mitochondria [33]. Severe ER stress, however, induces apoptosis by elevating levels of mitochondrial Ca 2+ and ROS, which either directly open the mitochondrial membrane permeability pore or indirectly promote BAX polymerisation. Under such conditions, mitochondrial survival is also regulated by the expression levels of anti-apoptotic Bcl-2 family proteins and by metabolic adaptation to aerobic glycolysis within the cytosol [21,[28][29][30][31][32][33][34][35].
Malignant tumours, including NB, are characterised by a glycolytic metabolic adaptation termed the "Warburg effect" [36,37]. This effect, not only provides a selective advantage for tumour cells by increasing glucose uptake to provide carbons for biosynthetic pathways but also promotes micro-environmental stress by increasing the extracellular concentration of lactate, resulting in a reductive acidic microenvironment. Maintenance of this microenvironment further selects stress-resistant tumour cells, is toxic for normal cells and facilitates formation of the cancer stem cell niche required for metastatic progression [38][39][40][41][42].
A greater understanding of the molecular mechanisms through which malignant tumours promote and maintain the "Warburg effect" should provide novel therapeutic ways to reverse its effect and slow tumour progression, as illustrated by metastasis suppressor KISS1 reversal of the Warburg effect [42]. Within this context, we present evidence for a novel stress-induced "Warburg"-promoting role for the TrkAIII oncoprotein in NB cells. We report that TrkAIII signals ER stress to the mitochondria, resulting in glycolytic metabolic adaptation, characterising a novel drug-reversible mechanism through which stress within the tumour microenvironment may maintain the "Warburg effect" in TrkAIII expressing NBs.

Mitochondrial TrkAIII targeting is enhanced under conditions of ER stress
In IF studies, TrkAIII co-localisation with MitoTracker Red-labelled mitochondria in non-stressed TrkAIII SH-SY5Y cells was significantly enhanced following treatment with DTT (5 mM), A23187 (10 µM) and thapsigargin (10 ng/ml) for 3 hours, from a mean (± s.e.) of 32.6 ± 2.1% in non-stressed TrkAIII SH-SY5Y cells, not significantly reduced by pre-incubation with the TrkA inhibitor CEP-701 (100 nM for 3 hours) [43], to 66.2 ± 2.15% following treatment with DTT, 62.7 ± 2.4% following treatment with A23187 and 51.2 ± 1.7% following treatment with thapsigargin (p < 0.0001, df = 98, for all three treatments) (Figure 2A), indicating that ER stress promotes TrkAIII targeting to the mitochondria. www.impactjournals.com/oncotarget TrkAIII localisation to mitochondria was confirmed by Western blot in density gradient ultracentrifugation purified mitochondria from non-stressed TrkAIII SH-SY5Y cells. Ultracentrifugation purified mitochondria were positive for TrkAIII and the mitochondrial markers TOM-20, Hsp60, PDHK1, cytochrome C and SOD2 but negative for the cytosolic proteins XIAP and α-tubulin and the ER protein calnexin ( Figure 2B). TrkAIII was detected in whole cell extracts (CELL EXT) positive for all markers and also in nuclei and mitochondria depleted extracts (MITO-free) positive for XIAP, calnexin and α-tubulin and depleted of SOD-2, Hsp60 and TOM20 ( Figure 2B). TrkAIII was constitutively Y490 and Y674/5 phosphorylated in whole cell (CELL EXT) and mitochondrial-depleted (MITO-free) extracts and but was not phosphorylated in mitochondrial extracts ( Figure 2B, data shown for TrkAIII Y674/5 phosphorylation only).
In addition to density gradient ultracentrifugationpurified mitochondria, TrkAIII was also detected in purified ER membranes, confirming previous reports [3,11], and in density gradient ultracentrifugation-purified MAMs but was not detected in membrane-free 100,000 x g ultracentrifugation cytosol fractions ( Figure 2C). TrkAIII positive MAMs were positive for TrkAIII, calnexin and TOM20 but not α-tubulin, whereas ER membranes were positive for TrkAIII and calnexin but not TOM20 and α-tubulin, confirming MAM purification as previously reported [44,45]. TrkAIII positive mitochondria were positive for TOM20 but not calnexin and α-tubulin and membrane-free cytosol was positive for α-tubulin but negative for TrkAIII, TOM20 and calnexin ( Figure 2C), indicating that TrkAIII localises to ER membranes, MAMs and mitochondria but not membrane-free cytosol.
OMM localisation for TrkAIII under non-stress conditions ( Figure 5A). Mitochondria from non-stressed cells also contained 30-35 kDa CT TrkAIII fragments ( Figure 5A) that were not digested by proteinase K alone and were only degraded by proteinase K in the presence of IMM permeabilizing digitonin concentrations, suggesting a mitochondrial matrix localisation ( Figure 5C).
In the absence of digitonin, proteinase K also degraded 100kDa TrkAIII to 18kDa CT and 63kDa NT fragments in mitochondria purified from DTT-treated TrkAIII SH-SY5Y cells but did not degrade the DTTinduced 48kDa CT Y674/5 phosphorylated TrkAIII cleavage-fragment or the constitutive 30-35kDa CT TrkAIII fragments ( Figure 5B and 5C). At OMM permeabilizing digitonin concentrations, proteinase K did not completely degrade 100kDa TrkAIII ( Figure  5B and 5C), suggesting increased internalisation of uncleaved TrkAIII into IMM structures protected from degradation (i.e. cristae) and also failed to degrade either the DTT-induced 48kDa or constitutive 30-35kDa TrkAIII cleavage-fragments. These fragments were only degraded in the presence of IMM permeabilizing digitonin concentrations (0.5 μg/ml), suggesting predominant localisation within the mitochondrial matrix ( Figure   Figure 5: DTT-induces mitochondrial TrkAIII cleavage to an active IMM-associated 48kDa CT fragmented in Mitochondrial matrix orientation. (A) Western blots demonstrating proteinase K (Prot K) digestion of TrkAIII in mitochondria from non-stressed TrkAIII SH-SY5Y cells to predominant C-terminal 18kDa (CT TrkAIII) and N-terminal 63kDa (NT TrkAIII) fragments, compared to TrkAIII in non-digested mitochondria (20 μg/lane). (B) Western blots demonstrating proteinase K (Prot K) cleavage of full length TrkAIII to 18kDa CT (CT TrkAIII) and 63kDa NT fragments (NT TrkAIII) but not DTT-induced 48kDa or constitutive 30-35kDa CT TrkAIII cleavage fragments in mitochondrial purified from DTT-treated (5mM for 6 hours) TrkAIII SH-SY5Y cells. (C) Western blots demonstrating proteinase K (Prot K) digestion of full length TrkAIII (TrkAIII) and full-length tyrosine phosphorylated TrkAIII (pY674/5 TrkAIII) but not DTT-induced 48kDa or constitutive 30-35kDa CT TrkAIII cleavage fragments in mitochondria from DTT-treated TrkAIII SH-SY5Y cells in the absence of digitonin (0), increased proteinase K digestion of full length TrkAIII but not DTT-induced 48kDa, constitutive 30-35kDa and proteinase K generated 18kDa CT TrkAIII cleavage fragments and HSP60, in the presence of 0.1-0.2 μg/ml digitonin and complete proteinase K digestion of DTT-induced 48kDa, constitutive 30-35kDa CT TrkAIII cleavage fragments and HSP60 but not the 18kDa OMM-associated TrkAIII CT fragment, in the presence of 0.5 μg/ml digitonin. www.impactjournals.com/oncotarget 5C). Interestingly, digitonin did not promote further degradation of the 18kDa TrkAIII fragment generated by proteinase K. This suggests that this fragment, which contains proteinase K degradation sites, may be protected by insertion within the OMM bilayer.
In Western blots, mitochondria purified from DTTtreated TrkAIII SH-SY5Y cells exhibited marked increase in mitochondrial protein tyrosine phosphorylation that was not detected in mitochondria from cells pre-incubated with CEP-701 (100 nm for 3 hours) or in mitochondria from pcDNA SH-SY5Y cells ( Figure 8C). Western blots also detected tyrosine phosphorylated PDHK1 in mitochondria purified from DTT-treated TrkAIII SH-SY5Y cells but not in mitochondria from DTT-treated pcDNA SH-SY5Y cells or from DTT-treated TrkAIII SH-SY5Y and pcDNA SH-SY5Y cells pre-incubated with CEP-701 (100 nM for 3 hours) ( Figure 8D). These data implicate TrkAIII activity in the DTT-induced tyrosine phosphorylation of mitochondrial proteins and PDHK1.
In an in vitro tyrosine kinase assay, tyrosine phosphorylated TrkAIII immunoprecipitated from mitochondria from DTT-treated TrkAIII SH-SY5Y cells, tyrosine phosphorylated exogenous recombinant PDHK1 in vitro to a greater degree in the absence of CEP-701 compared to the presence of CEP-701 (100 nM) and also compared to TrkAIII immunoprecipitated from mitochondria from untreated TrkAIII SH-SY5Y cells ( Figure 8E). This suggests that PDHK1 is a novel TrkAIII substrate.

DTT, A23187 and thapsigargin promote glycolysis in TrkAIII SH-SY5Y cells
In glycolysis assays, lactate production integrated with growth over 24 hours did not significantly differ between non-stressed pcDNA SH-SY5Y and TrkAIII SH-SY5Y cells (P = 0.357 df = 10). DTT, A23187 and thapsigargin-treatment promoted extensive death of pcDNASH-SY5Y but not TrkAIII SH-SY5Y cells but did prevent TrkAIII SH-SY5Y growth over the 24-hour assay period, whereas non-stressed TrkAIII SH-SY5Y cells exhibited a growth rate of 3.8 ± 0.5% and more than doubled within 24 hours.

Figure 10: TrkAIII augments SH-SY5Y cell-resistance to DTT, A23187 and thapsigargin-induced death. (A) Micrographs
demonstrating increased death of pcDNA SH-SY5Y cells compared to TrkAIII SH-SY5Y cells (red = dead, green = alive), following treatment with DTT (5 mM for 6 hours), A23187 (A23, 10 µM for 6 hours) and thapsigargin (THA, 10 ng/ml for 6 hours) in 16 hour assays (bar=100 μm). Histograms displaying: (B) significant differences ( * ) in TrkAIII SH-SY5Y cell death compared to pcDNA SH-SY5Y cell death, following treatment with DTT (5 mM), A23187 (A23, 10 µM) and thapsigargin (THA, 10 ng/ml); (C) significant ( * ) increase in DTTinduced TrkAIII SH-SY5Y death in cells pre-incubated with CEP-701 (100 nM, CEP) but not in cells pre-incubated with Ucf-101 (UCF, metabolic adaptation. ER stress induced by DTT, A23187 and thapsigargin promoted TrkAIII targeting to the mitochondria and internalisation into IMMs, resulting in Omi/HtrA2-dependent cleavage-activation of TrkAIII to active CT-fragments in predominantly tyrosine kinase domain mitochondrial matrix orientation. We provide evidence that this process depends upon a stress-induced increase in ROS production underpinned by changes in Ca 2+ movement and that stress-induced cleavageactivation of mitochondrial TrkAIII results in the tyrosine phosphorylation of mitochondrial PDHK1, leading to a glycolytic metabolic adaptation. Finally, we show that the TrkAIII inhibitor CEP-701 and the Omi/HtrA2 inhibitor Ucf-101 prevent this mechanism. TrkAIII targeting to the mitochondria could not be explained simply on the basis of a NT mitochondrial translocation sequence, which was calculated online with a low probability (0.2) of mitochondrial translocation (http:// mitf.cbrc.jp/MitoFates/cgi-bin/top.cgi), implicating an atypical mechanism. Furthermore, mitochondrial TrkAIII was neither poly-mono-ubiquinated nor poly-ubiquitinated (data not shown), suggesting an alternative localisation mechanism to that reported for ubiquitinated p53 [55]. The TrkAIII inhibitor CEP-701 did not reduce mitochondrial TrkAIII levels, indicating a TrkAIII activity-independent mechanism and the Hsp90 inhibitor Geldanamycin-A also failed to reduce mitochondrial TrkAIII levels, indicating that Hsp90, which binds TrkAIII [3] and regulates mitochondrial protein importation through TOM-70 [56], was also not involved. Potential involvement of the ER chaperone Grp78/Bip, which also binds TrkAIII [3] and exhibits ER stress-induced mitochondrial translocation [57], is under investigation. Alternatively, TrkAIII may be recognised as a damaged or stress-protein and targeted to the mitochondria for degradation [58]. This possibility is supported by the detection of 30-35kDa CT TrkAIII degradation fragments in mitochondrial matrices under both non-stress and ER-stress conditions.
TrkAIII was not only detected in purified mitochondria but also in ER membranes and MAMs. TrkAIII in purified MAMs, positive for TOM20 and calnexin but not α-tubulin as previously reported [44,45], was not enriched compared to ER membranes and mitochondria, confirming its association with all three compartments [this study, 3,11]. The presence of TrkAIII in MAMs suggests that ER-associated TrkAIII may gain access to mitochondria via these sites.
Proteinase K digestion of mitochondrial TrkAIII to an 18kDa CT fragment, resistant to further degradation in the presence IMM permeabilizing digitonin concentrations, suggests that this hydrophobic domain of TrkAIII [59] may be anchored and inserted within the OMM bilayer, protecting it from further degradation.
In contrast to non-mitochondrial TrkAIII, mitochondrial TrkAIII was not constitutively Y490 or Y674/5 tyrosine phosphorylated under non-stress conditions. This indicates that mitochondria have a higher threshold for spontaneous TrkAIII activation than either ERGIC/COP1 membranes or the centrosome [1,11,12]. A potential role for mitochondrial PTPases in maintaining this high threshold was supported by detection of the TrkA-de-phosphorylating PTPases SHP-2 and PTP1B [60,61] in purified mitochondria and by rapid activation of mitochondrial TrkAIII by the PTPase inhibitors sodium orthovanadate and H 2 O 2 [62,63]. NGF, on the other hand, could not activate mitochondrial TrkAIII.
Treatment of TrkAIII SH-SY5Y cells with the ER stress inducers DTT, A23187 and thapsigargin activated the ERSR, increased TrkAIII targeting to mitochondria and promoted the mitochondrial TrkAIII internalisation into IMMs. Mitochondrial TrkAIII targeting may involve Grp78/bip, which binds TrkAIII [3] and exhibits ER stress-induced translocation from the ER to mitochondria [57]. Stress-induced mitochondrial TrkAIII internalisation, on the other hand, may involve Omi/HtrA2, since the Omi/HtrA2 inhibitor Ucf-101 [46] reduced mitochondrial TrkAIII levels under conditions of ER stress. We are further investigating these possibilities.
Stress-induced internalisation of mitochondrial TrkAIII into IMMs was associated with TrkAIII cleavage to 45-48kDa active Y490 and Y674/5 phosphorylated CT fragments in tyrosine kinase domain mitochondrial matrix orientation, suggesting that TrkAIII signals ER stress to the mitochondrial matrix. Focussing upon the effect of DTT, mitochondrial TrkAIII cleavage-activation was prevented by both Ucf-101 and siRNA Omi/HtrA2 knockdown, implicating Omi/HtrA2. At present, it remains unclear whether Omi/HtrA2 degrades TrkAIII directly or indirectly via activation of other enzymes. Furthermore, A23187 induced mitochondrial TrkAIII cleavage to a 45kDa active CT fragment rather than the 48kDa active CT fragments induced by DTT and thapsigargin, suggesting involvement of additional and/or alternative proteases. A23187 also reduced the size of full-length mitochondrial TrkAIII to 95kDa, detected by an antibody against the TrkA CT-terminus, suggesting that it may promote additional cleavage at the TrkAIII CT terminus. Ucf-101, however, abrogated TrkAIII activation by all three agents, supporting a common role for Omi/HtrA2. In contrast to Omi/HtrA2, we found no evidence for the involvement of caspases, MMPs or ADAMS in DTT-induced cleavagedependent mitochondrial TrkAIII activation. Together, these data suggest that Omi/HtrA2 plays a central role in stress-induced mitochondrial TrkAIII cleavage-activation, providing a novel addition to ADAMs/secretase-dependent cleavage-activation of fully-spliced cell surface TrkA [64]. Furthermore, Ucf-101 not only abrogated DTT-induced TrkAIII cleavage but also phosphorylation, implying a cleavage-dependent activation mechanism, presumably facilitated by the elimination of remaining spontaneous activation-prevention domains from TrkAIII [9]. This unveils a novel role for mitochondrial Omi/HtrA2 in stress-protection [31,32]. Whether this mechanism represents a ubiquitous response to ER stress, remains to be fully established. TrkAIII cleavage-activation was induced to varying degrees by other ER stress inducers such as brefeldin A, 2-deoxyglucose and DDT-ox but was not induced by the N-glycosylation blocker tunicamycin (data not shown), suggesting that this mechanism is not entirely ubiquitous. However in contrast to the other agents, tunicamycin promoted TrkAIII de-glycosylation to a 70kDa core protein that remained trapped within the ER (data not shown), which may explain this observation. Furthermore, although tunicamycin promotes Omi/HtrA2 expression [65], its effects upon Omi/HtrA2 activity have not yet been reported.
ROS involvement in cleavage-dependent mitochondrial TrkAIII activation was suggested by increased ROS production induced by DTT, A23187 and thapsigargin, and supported using the anti-oxidant Resveratrol [48], which prevented DTT, A23187 and thapsigargin stimulation of ROS production and abrogated stress-induced mitochondrial TrkAIII cleavage-activation. The observation that H 2 O 2 activated mitochondrial TrkAIII, suggests that the increase in ROS production induced by ER stress facilitates mitochondrial TrkAIII cleavage-activation by inactivating mitochondrial PTPases. Furthermore, increased mitochondrial ROS production has been reported to activate Mpv17l, an IMMassociated Omi/HtrA2 activator [32].
A Ca 2+ ionophore (A23187) [49], a SERCA Ca 2+ pump inhibitor (thapsigargin) [50] and a ryanodine receptor Ca 2+ channel agonist (DTT) [51] all induced mitochondrial TrkAIII cleavage-activation, suggesting a central role for Ca 2+ in this process. The ryanodine receptor Ca 2+ channel inhibitor dantrolene [52] abrogated DTT-induced cleavage and phosphorylation of mitochondrial TrkAIII, indicating that the DTT-effect was ryanodine receptor Ca 2+ channeldependent. Unfortunately, we were unable to sufficiently knockdown ryanodine receptor expression to confirm this. In contrast, dantrolene did not inhibit either A23187 or thapsigargin-induced mitochondrial TrkAIII cleavageactivation, consistent with the ryanodine receptorindependent Ca 2+ mobilising mechanisms of these agents [49,50]. The IP3R Ca 2+ channel inhibitor 2APB [53] also failed to inhibit DTT, A23187 or thapsigargin-induced mitochondrial TrkAIII cleavage-activation, confirming an IP3R-independent mechanism for all three agents. Therefore: ER release of Ca 2+ via ryanodine receptors; inhibition of ER Ca 2+ replenishment and receptor/channelindependent alterations in Ca 2+ compartmentalisation, are independently capable of inducing mitochondrial TrkAIII cleavage-activation. Furthermore, increased mitochondrial Ca 2+ levels have been shown to activate Omi/HtrA2 [66] and augment ROS production [21,23,26]. This suggests that Ca 2+ /ROS interplay provides conditions necessary to overcome the mitochondrial TrkAIII activation threshold by activating Omi/HtrA2, cleaving TrkAIII and inhibiting PTPase activity. We are further investigating the molecular pathways involved.
Stress-induced mitochondrial TrkAIII cleavageactivation resulted in the tyrosine phosphorylation of mitochondrial proteins, including PDHK1. Stress-activated mitochondrial TrkAIII tyrosine phosphorylated exogenous recombinant PDHK1 in vitro and CEP-701 prevented stress-induced PDHK1 tyrosine phosphorylation and the phosphorylation of recombinant PDHK1 by TrkAIII in vitro, characterising PDHK1 as a novel potential TrkAIII substrate. PDHK1 tyrosine phosphorylation has been reported to inhibit the pyruvate dehydrogenase complex (PDC), promote lactate production and induce aerobic glycolysis [67,68]. Consistent with this, DTT, A23187 and thapsigargin increased lactate production and glucose uptake in TrkAIII SH-SY5Y, indicating a metabolic shift to aerobic glycolysis. This was not observed in control pcDNA SH-SY5Y cells and was prevented by CEP-701 and Ucf-101, implicating both TrkAIII and Omi/HtrA2 activity.
ER stress induced glycolytic adaptation in TrkAIII SH-SY5Y cells was also associated with enhanced resistance to DTT, A23187 and thapsigargin-induced death. CEP-701 increased the sensitivity of TrkAIII SH-SY5Y cells ER stress-induced death, implicating TrkAIII activity in enhancing survival. This adds to previous reports that TrkAIII increases resistance of NB cells to a variety of toxic agents [1,3,7]. The induction of caspase-dependent apoptosis in DTT-treated pcDNA SH-SY5Y cells was prevented by z-VAD-fmk and associated with caspase-9 and caspase 3 cleavage, implicating the intrinsic apoptosis pathway and suggesting that TrkAIII must block this pathway. In support of this, TrkAIII SH-SY5Y cells exhibit elevated expression of the intrinsic apoptosis inhibitors Bcl-2, Bcl-xL and Mcl-1 and TrkAIII inhibition reduced Bcl2 and Bcl-xL expression [54]. Here, we report that TrkAIII SH-SY5Y mitochondria contained higher levels of Bcl-2, Bcl-xL and Mcl-1 proteins, providing an explanation for the increased resistance to apoptosis mediated via the intrinsic pathway and increased sensitivity to ER stress-induced death in the presence of CEP-701. In contrast, Ucf-101 did not increase TrkAIII SH-SY5Y sensitivity to ER stress-induced death nor reduce Mcl-1, Bcl-2 or Bcl-xL expression, suggesting that extra-mitochondrial rather than mitochondrial TrkAIII activity is responsible for enhancing Bcl-2 and Bcl-xL expression and survival of TrkAIII SH-SY5Y cells, under conditions of ER stress.
In conclusion, we have identified a novel role for the TrkAIII oncoprotein in signaling ER stress to the mitochondria in NB cells that results in glycolytic metabolic adaptation and associates with enhanced survival. We propose that ER stress promotes TrkAIII targeting to the mitochondria and its internalisation into IMMs. This results in Omi/HtrA2-dependent TrkAIII cleavage-activation that also depends upon Ca 2+ /ROS interplay, which provides the conditions necessary to overcome the mitochondrial TrkAIII activation threshold by activating mitochondrial Omi/HtrA2 and inhibiting mitochondrial PTPases. The activation of this mechanism results in tyrosine phosphorylation of mitochondrial PDHK1, leading to a glycolytic metabolic adaptation. We propose that this represents a novel self-perpetuating, drug-reversible, mechanism through which tumour microenvironmental stress may maintain the metastasis promoting "Warburg effect" in TrkAIII expressing NBs. TrkAIII SH-SY5Y and pcDNA SH-SY5Y cell lines were obtained from SH-SY5Y neuroblastoma cells (ATCC CRL2266), have been described previously [1] and were routinely cultured in DMEM supplemented with 10% foetal calf serum, glutamine and antibiotics (penicillin, streptomycin for all cultures plus Zeocin).

Purification of mitochondria, mitoplasts and MAMs
Mitochondria were purified using a Focus Sub cell Mitochondrial isolation kit, as described by the manufacturer (G-Biosciences, St. Louis, MO). Briefly, cells were harvested in ice cold PBS by scraping, centrifuged at 1,200 × g for 5 minutes at 4°C, 500 μl of ice cold Buffer I, containing 1× protease inhibitor cocktail (Sigma) was added to the pellet and cells were disrupted by 10 passages through a 20 gauge needle. 250 μl of Buffer II was then added to the homogenate and samples were then centrifuged at 1,200 × g for 5 minutes, at 4°C. Supernatants were transferred to fresh tubes, centrifuged at 15,000 x g for 10 minutes at 4°C and the resulting mitochondrial-rich pellet was washed with 500 μl of Buffer II, centrifuged at 15,000 × g for 10 minutes at 4°C and re-suspended in mitochondrial storage buffer (250mM mannitol, 5-mM HEPES (pH 7.4). Crude mitochondrial preparations in 250mM mannitol, 5mM HEPES (pH 7.4) and 0.5mM EGTA were then separated by Percoll density gradient centrifugation, as previously described [69]. Briefly, crude mitochondrial preparations were layered onto a 30% Percoll gradient in the same buffer and ultra centrifuged at 90,000 × g for 40 minutes at 4°C. Ultrapure mitochondria were collected from a band located 2/3rds from the top, transferred to a fresh tube, diluted 1:10 in 250 mM mannitol, 5 mM HEPES (pH 7.4) containing 0.5 mM EGTA and re-centrifuged at 15,000 × g for 10 minutes, at 4°C. Mitochondrial pellets were then re-suspended in the desired volume of cold 250 mM mannitol, 5-mM HEPES (pH 7.4) containing 0.5 mM EGTA immediately prior to experimentation.
Mitoplasts were obtained from purified mitochondria as previously described [70]. Briefly, ultra purified mitochondria were incubated with 0.1 μg digitonin/ml of mitochondrial protein with gentle stirring on ice for 15 minutes. Three volumes of 250 mM sucrose in mannitol/HEPES/EGTA pH [7.4] buffer were then added. Mitoplasts were pelleted by centrifugation at 15,000 × g for 10 minutes and re-suspended in SDS PAGE reducing sample buffer.
MAMs were purified as previously described [43]. Briefly, cells (10 9 ) were harvested, washed in PBS, pelleted at 500 × g for 5 min, re-suspended in homogenization buffer (0.25 M sucrose and 10 mM HEPES pH 7.4) and dounce-homogenized. Homogenates were centrifuged twice at 600 × g for 5 min to remove cellular debris and nuclei, and supernatants centrifuged at 10,300 × g for 10 min to pellet crude mitochondria. Mitochondria, re-suspended in 250 mM mannitol, 5 mM HEPES (pH7.4), and 0.5 mM EGTA, were layered on top of 8 ml of Percoll medium (225 mM mannitol, 25 mM HEPES (pH 7.4), 1 mM EGTA and 30% Percoll (v/v)) in a 10-ml polycarbonate ultracentrifuge tube and centrifuged for 30 min at 95,000 × g. MAMs, removed as a diffuse white band above mitochondria, were diluted in isolation medium, centrifuged at 6,300 × g for 10 min and the supernatant containing MAMs was centrifuged at 100,000 × g for 1h in a Beckman 70 Ti rotor. The resulting MAM pellet was re-suspended in homogenisation buffer, protein concentrations measured and examined by reducing SDS PAGE Western blotting. Purified MAMs were positive for calnexin and TOM20 but negative for α-tubulin [44,45], ER fractions were positive for calnexin but negative for TOM20 and α-tubulin, mitochondrial fractions were positive for TOM20 but negative for calnexin and α-tubulin and membrane-free cytosol fractions prepared by 100,000 × g ultracentrifugation, were negative for calnexin and TOM20 but positive for α-tubulin. For proteinase K digestion, purified mitochondria or mitoplasts were incubated with Proteinase K (10 μg/ml) at 0°C for 30 minutes in the presence or absence of digitonin (0.1-0.5 μg/ml), reactions were stopped with 2mM PMSF, digested mitochondria and mitoplasts were pelleted by centrifugation at 15,000 × g for 10 minutes at 4°C, resuspended in reducing SDS-PAGE sample buffer and analysed by Western blot.

Labeling mitochondria with MitoTraker-Red and MitoSox-Red
Cells grown on glass chamber slide (Nunc) were treated with reagents at concentrations and for the times indicated in the text and figure legends. Following incubation, cells were washed 5 times with pre-warmed PBS (37°C), particularly important for DTT which inhibits reagent fluorescence, and either MitoTraker-red (for mitochondria localization) or Mitosox red (for ROS detection) solution added to a final concentration of 2.5μM, and incubated for 15 minutes at 37°C. Cells were then washed three times in pre-warmed PBS, mounted in PBS/Glycerol and examined immediately under a Zeiss "Axioplan-2" fluorescence microscope. Representative fields were digitally photographed under identical exposure conditions.

Inhibitor studies
In inhibitor studies, cells were pre-incubated with aprotinin, z-VAD-fmk, TAPI-2, EDTA, CEP-701 and Ucf-101 in complete medium for 3 hours prior to the addition of DTT, A23187 or thapsigargin and inhibitors were also present for the duration of incubation with these agents. For Resveratrol studies, cells were pre-incubated for 12 hours with Resveratrol prior to the addition of stress-inducing agents and for the duration of subsequent incubation.

Immunoprecipitation and Western blotting
Cells or purified mitochondria were extracted in lysis buffer (PBS containing 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 1mM sodium orthovanadate, 1mM PMSF, 1 μg/ml of pepstatin A and Aprotinin) and protein concentrations calculated by Bradford protein concentration assay (Sigma-Aldrich). Samples for SDS-PAGE were mixed with reducing SDS-PAGE sample buffer and subjected to reducing SDS-PAGE/ Western blotting. Briefly, proteins separated by reducing SDS-PAGE, were trans-blotted onto Hybond C+ nitrocellulose membranes by electrophoresis (Amersham Int. UK) and the membranes subsequently air-dried. Non-specific protein binding-site on membranes were blocked by incubation for 2 hours in 5% non-fat milk in TBS-T prior to incubation with primary antibodies, at recommended dilutions, for 2-16 hours at 4°C. Membranes were then washed in TBS-T, incubated with secondary HRP-conjugated antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in blocking solution and immunoreactive species detected by chemiluminescence reaction, as directed by the manufacturer (Amersham Int). For immunoprecipitation, extracts (200-500 μg) pre-cleared with IgG and protein sepharose A were incubated with primary antibody at a concentration of 0.1-1.0 μg/500 mg total protein for 2-16 hours at 4°C. Following incubation, 20 μl of Protein A Sepharose (Fast flow, Sigma-Aldrich) in lysis buffer, was added and incubated for 30 minutes at 4°C, with rotation. Protein sepharose/IgG conjugates were then collected by centrifugation (10,000 × g for 5 minutes), washed 3 times in lysis buffer, re-suspended in SDS-PAGE sample buffer and subjected to reducing SDS-PAGE/Western blotting.

Cell death assay
Cell death assays were performed as previously described [7]. Briefly, cell cultures were washed once in Ca 2+ free PBS, detached with ice cold PBS containing 1mM EDTA, transferred to sterile 15 mls tubes, centrifuged for 5 minutes at 1,000× g at 4°C, washed again in ice cold PBS and re-pelleted by centrifugation at 1,000 × g for 5 minutes at 4°C. Cell pellets were re-suspended in 25 μl of PBS containing 2 μl of acridine orange/ethidium bromide solution (100 μg/ml acradine orange and 100 μg/ml ethidium bromide in PBS) plated onto glass slides and examined immediately under a Zeiss "Axioplan-2" fluorescence microscope. Representative fields were digitally photographed under identical exposure conditions and the number of dead cells (orange/red nuclei) and live cells (green nuclei) counted.

Indirect immunofluorescence
Cells grown on Nunc glass chamber slides (Sigma-Aldrich) were washed in PBS, fixed and permeabilized in 100% ice cold methanol (-20°C), washed in PBS then processed for indirect immunofluorescence (IF). Fixed, permeabilized cells were incubated for 1 h in blocking solution (1% bovine serum albumin in PBS-0.03% TX100) and then incubated for 2 hours with primary antibody in blocking solution, at room temperature.
Slides were then washed three times in PBS-0.03% TX100, incubated with secondary fluorochromeconjugated antibody diluted in blocking solution for 1 h at room temperature, washed in PBS-0.03% TX100 and mounted using VectorMount TM , containing DAPI nuclear counterstain. Images were obtained using a Zeiss Axioplan 2 fluorescence microscope with digital camera and Leica M500 Image Manager software.

Glycolysis assay
Glycolysis was measured by L-Lactate production using a cell-based glycolysis assay kit, as described by the manufacturer (Cayman chemical). Briefly, 1 × 10 5 cells per well were plated onto 96 well plates for glycolysis assays and parallel cell counting, and grown for 12 hours. At 12 hours, cell counts were performed on parallel cultures and duplicate experimental cultures (and parallel cell count cultures) incubated with reagents (medium alone, DTT, A23187 or thapsigargin with or without pre-incubation with inhibitors at the concentrations and times indicated in the text, in a volume of 100 ml complete culture medium, for 6 hours at 37°C. At 6 hours, cell counts were performed on parallel cultures, and in experimental cultures supernatants were removed, cultures were washed 5 times in 150 ml of pre-warmed PBS, 100 ml of fresh medium (containing 1%FCS) added to each well and then incubated for 24 hours at 37°C. At 24 hours, cells were counted and supernatants were removed, centrifuges at 14,000 rpm to remove cell debris and L-Lactate levels were measured in duplicate 10 ml aliquots of culture supernatant and compared to an L-lactate standard curve, as directed by the manufacturer (Cayman Chemicals). Exponential growth rates were calculated from cell counts using the online exponential growth rate calculator at http://www.rapidtables.com/ calc/math/ exponential-growth-calculator.htm. Growth rates (units per hour) were used to calculate lactate production rates using the general equation: Production Rate = m(Cf Ci) / (Df Di), where m = growth rate, Cf = final lactate concentration, Ci = the initial Lactate concentration, Df = final cell density and Di = initial cell density [71]. Lactate production rates were calculated as M lactate/ 100 cells per hour and data presented as fold difference (± s.e.) compared to untreated controls.

Glucose uptake assays
Glucose uptake was measured using a cell-based glucose assay kit, as described by the manufacturer (Cayman chemical). Briefly, cells at a concentration of 5 × 10 4 cells/well were seeded onto 96 well plates (for experiments and parallel cell counts), grown overnight then treated for 6 hours with DTT, A23187 or thapsigargin with or without 2 hour pre-incubation with CEP-701 or Ucf-101, in a volume of 100 μl glucose-free culture medium. 2-NBDG (150 μg/ml) was then added in glucosefree medium and cultures incubated for a further 1 hour. At 1 hour, cultures were centrifuged at 400 × g for 10 minutes at RT, supernatants aspirated, 200ml of cell-based assay buffer added, plates re-centrifuged at 400 × g for 5 minutes at RT, supernatants aspirated, 100 μl of cell-based assay buffer added to each well and plates read promptly in a fluorescence plate reader (excitation/emission = 485/535 nm). Serial dilutions of a 1 mg/ml stock solution of 2-NBDG in assay buffer were used as a reference standard. Fluorescent measurements were adjusted for cell numbers, counted using the phase contrast micrographs prepared immediately prior to fluorescence analysis and are presented as fold difference (± s.e.) to untreated controls. 2-NBDG up-take was also examined by direct fluorescence microscopy in cells cultured on Nunc chamber slides, following identical treatment to that used in glucose uptake assays. At the termination of treatment, cells were washed in pre-warmed PBS, mounted in PBS/ Glycerol, visualised under a Zeiss Axioplan 2 fluorescence microscope with digital camera and Leica M500 Image Manager software and light micrographs taken.

Densitometry, statistical analysis and software
Data were analysed statistically using the Student's t-test, using the online t-test calculator at https://www. graphpad.com/quickcalcs/ttest1.cfm, with statistical significance associated with probabilities of ≤ 0.05. Mitochondrial translocation probability was calculated using the online protein N-terminal mitochondrial translocation probability calculator at http://mitf.cbrc.jp/ MitoFates/cgi-bin/top.cgi. Exponential growth rates were calculated from cell counts, using the online calculator at http://www.rapidtables.com/calc/math/ exponentialgrowth-calculator.htm. Densitometric and quantification of co-localisation analyses were performed using Image J Fiji software, with colour pixel counter plugin [72].


text:
NUMBL Interacts with TAK1, TRAF6 and NEMO to Negatively Regulate NF-κB Signaling During Osteoclastogenesis

NF-κB signaling is essential for osteoclast differentiation and skeletal homeostasis. We have reported recently that NUMB-like (NUMBL) protein modulates osteoclastogenesis by down regulating NF-κB activation. Herein, we decipher the mechanism underlying this phenomenon. We found that whereas NUMBL mRNA expression decreases upon stimulation of wild type (WT) bone marrow macrophages (BMMs) with RANKL, TAK1 deficiency in these cells leads to increased NUMBL and decreased TRAF6 and NEMO expression. These changes were restored upon WT-TAK1 expression, but not with catalytically inactive TAK1-K63W, suggesting that TAK1 enzymatic activity is required for these events. Forced expression of NUMBL inhibits osteoclast differentiation and function as evident by reduction in all hallmarks of osteoclastogenesis. Conversely, NUMBL-null BMMs, show increased osteoclast differentiation and mRNA expression of osteoclast marker genes. Post-translationally, K48-linked poly-ubiquitination of NUMBL is diminished in TAK1-null BMMs compared to elevated K48-poly-ubiquitination in WT cells, indicating increased stability of NUMBL in TAK1-null conditions. Further, our studies show that NUMBL directly interacts with TRAF6 and NEMO, and induces their K48-poly-ubiquitination mediated proteasomal degradation. Collectively, our data suggest that NUMBL and TAK1 are reciprocally regulated and that NUMBL acts as an endogenous regulator of NF-κB signaling and osteoclastogenesis by targeting the TAK1-TRAF6-NEMO axis.

modulate NF-κB activity in health and disease. To this end, various regulators that interact with and regulate NF-κB signaling during basal and pathologic osteoclastogenesis have been identified.
TAK1 activates NF-κB through phosphorylation of IKK2 following stimulation with various stimuli, including RANKL, IL-1β and TNFα. The process involves recruitment of TAK1 to the NF-κB signalosome through adaptor proteins, such as TAB2 and polyubiquitin chains that facilitate arrangement of signaling proteins including TRAF6, NEMO, at close proximity. Using gene deletion studies, we have shown recently that TAK1 and NEMO are both essential for NF-κB activation and osteoclastogenesis, as absence of either protein enabled osteopetrosis 5,7 . Mechanistically, we have shown that TAK1 regulates expression of NUMB-like (NUMBL) and subsequently interact with Notch intracellular domain (NICD)/RBPJ signaling pathway during OC differentiation 7 .
NUMB and NUMBL have been studied as evolutionary conserved proteins which play a role in cellular fate determination during development 22,23 . However, the role of these proteins as TAK1 targets and regulators of NF-κB is poorly understood and their role in osteoclastogenesis is unknown. To this end, we have shown that deletion of TAK1 is associated with simultaneous increased expression of NUMBL and decreased expression of TAK1 adaptor protein TAB2. The increased NUMBL expression induces degradation of NICD, resulting in accumulation of co-repressor RBPJ that blunts NFATc1 expression and OC differentiation 7 . However, the interaction of NUMBL with TAK1/TAB2 and other components of NF-κB signaling during OC differentiation have not been fully elucidated.
In this regard, transfection studies have pointed to possible regulation of NF-κB signaling through interaction of NUMBL with TAB2 and TRAF6 in neurons 24,25 . Another report suggests that NUMBL regulates glioma cells migration and invasion by inhibiting TRAF5-induced NF-κB activation 26 . Based on these reports and our finding that NUMBL is involved in OC differentiation 7 , we herein studied the mechanism of NUMBL mediated regulation of NF-κB signaling and OC differentiation.
We found that during osteoclastogenesis, NUMBL expression is regulated at the transcriptional and translational levels. Our data indicate that RANKL stimulation decreases NUMBL mRNA expression in TAK1 sufficient bone marrow cells. It further suggests that TAK1/TAB2 complex mediates poly-ubiquitination of NUMBL marking it for proteasomal degradation. We also found that increased NUMBL expression leads to a concomitant decrease in TRAF6 and NEMO expression which results in inhibition of NF-κB activation and reduced OC differentiation and function. In summary, our data establishes NUMBL as a negative regulator of NF-κB signaling acting via interaction with TRAF6-TAB2-TAK1 complex and leading to regulation of the NICD-RBPJ axis, as we have shown recently 7 . Additional studies investigating the transcriptional control of NUMBL during OC differentiation will provide further understanding the mechanism of role of NUMBL in regulating bone homeostasis.

Results
TAK1 deletion leads to increased expression of NUMBL and decreased expression of TRAF6 and NEMO. We have shown previously that conditional deletion of TAK1 in the myeloid lineage plummets RANKL-induced OC differentiation and that this process is associated with elevated expression of NUMBL concurrent with reduced NF-κB activation 7 . We also showed that combined deletion of NUMB/NUMBL form TAK1-cKO mice partially restores the osteopetrotic phenotype of TAK1-null mice and osteoclastogenesis 7 . Here we examined the possibility that NUMBL plays a repressor role during osteoclastogenesis. Specifically, we examined a potential mechanistic correlation between NUMBL expression and the key OC differentiation pathway encompassing TRAF6 and downstream NF-κB molecules. To this end, wild type (WT) and TAK1-null bone marrow cells were isolated and treated with vehicle, RANKL and/or TNF-α as indicated in Fig. 1A and B. Expression of TRAF6 and NEMO is stable in WT cells, but is significantly reduced in TAK1-null cells when equal total protein is loaded from the various conditions (confirmed by β-actin expression) ( Fig. 1A and B). In contrast, expression of NUMBL is highly elevated in TAK1-null cells (Fig. 1A). This observation suggests that the divergent expression of NUMBL compared with TRAF6 and NEMO intersects at TAK1, and TAK1 not only mediates expression of TRAF6 and NEMO, but also regulates expression of potential repressors such as NUMBL.

Expression of pMX-TAK1 inhibits NUMBL and restores TRAF6 and NEMO expression.
To experimentally confirm that TAK1 indeed negatively regulates NUMBL, we examined the effect of forced expression of retroviral TAK1 in bone marrow cells. Our previous data show that WT (pMX-TAK1-WT) but not catalytically inactive TAK1 (pMX-TAK1-K63W) inhibits NUMBL expression concurrent with increased expression of TAB2 in in TAK1 null cells 7 . Under similar experimental conditions we observed that TAK1-WT but not TAK1-K63W rescues TRAF6 and NEMO expression in TAK1 null cells (Fig. 1C). These observations suggest that NUMBL directly or indirectly suppresses TRAF6 and NEMO expression and that TAK1 inhibits this action. More importantly, the data point out that NUMBL-induced repression of TRAF6 and NEMO in TAK1-null cells is reversible upon reconstitution of active TAK1 expression.
NUBML acts as a negative regulator of osteoclastogenesis. Our data suggest that TAK1 deletion leads to increased NUMBL expression with concomitant decrease in TRAF6 and NEMO expression. Based on this finding, we hypothesized that NUMBL acts as a negative regulator of osteoclastogenesis. To test this proposition, WT bone marrow cells were transfected with pMX-GFP and pMX-NUMBL and subjected to osteoclastogenesis, actin ring formation, as well as qPCR and biochemical analyses. Our data show that NUMBL overexpression decreases RANKL induced OC differentiation ( Fig. 2A,B) and fewer actin rings on bone slices (Fig. 2C). Further, qPCR data show that NUMBL over-expression results in decreased mRNA expression of TRAP, NFATc1, DC-STAMP, MMP9, β3-integrin and cathepsin-K. (Fig. 2D,J). Western blot analysis also shows a decrease in NFATc1 expression in response to RANKL treatment of pMX-NUMBL overexpressing cells (Fig. 2K).
To confirm the effect of increased NUMBL expression on NF-κB activation, RelA-Luc reporter assay was performed using bone marrow cells isolated from RelA_luc-reporter mice. The luminescence activity shows that NUMBL expression decreases NF-κB activation in response to RANKL treatment when compared with control GFP expressing cells (Fig. 2L). Expression of transduced pMX-flag-NUMBL was confirmed by western blot using FLAG antibody (Fig. 2M). This data suggest that increased NUMBL expression inhibits OC differentiation.

Deletion of NUMBL in myeloid lineage leads to increase in osteoclastogenesis.
To further investigate the effect of NUMBL on basal OC differentiation, bone marrow cells from WT and Numb/Numbl double knockouts (N/NL-dcKO) mice (as detailed in the methods section) were differentiated to osteoclasts in the presence of RANKL and M-CSF for 4 days. We decided to use cells from mice lacking both NUMB and NUMBL to avoid any potential redundancy of NUMB. The results indicate that deletion of NUMBL leads to increased OC differentiation (Fig. 3A,B). This observation is further supported by increased mRNA expression of OC differentiation associated genes TRAP, NFATc1, DC-STAMP, β3-integrin, cathepsin-K and MMP9 ( Fig. 3C-H).

RANKL inhibits NUMBL expression and induces NUMBL K48-linked poly UB in TAK1-dependent manner.
Based on our data, we next studied potential mechanisms underlying TAK1-dependent regulation of NUMBL during RANKL-induced OC differentiation. To understand the mechanism of NUMBL inhibition, we measured the NUMBL mRNA and protein expression in RANKL stimulated bone marrow cells. Contrary to increases observed in NUMBL protein expression after 3-4 days of RANKL treatment (Fig. 1A), WT bone marrow cells treated with RANKL for 1 day in the presence of M-CSF show decrease in NUMBL mRNA expression (Fig. 4A). In contrast, in TAK1-null cells, RANKL stimulation failed to inhibit NUMBL mRNA expression (Fig. 4A). Next, to investigate if TAK1 regulates NUMBL expression post-translationally, we measured ubiquitination of NUMBL in immunoprecipitates from vehicle or RANKL-treated WT and TAK1cKO-derived bone marrow cell lysates. The data suggest that TAK1 deletion leads to hypo-K48-linked poly UB of NUMBL, which stabilizes its expression. These observations along with preivious data showing decreasesed NUMBL and increased TAB2 expression in TAK1-null expressing TAK1-WT but not catalytically inactive TAK1-K63W 7 , suggest that under normal conditions, TAK1 activity and presence of TAK1-TAB2 signaling may mediate poly-ubiquitination of NUMBL leading to its proteasomal degradation (Fig. 4B).

NUMBL overexpression decreases TRAF6 and NEMO abundance by inducing their K48-linked poly-Ubiquitination.
Our results indicate that RANKL-induced NF-κB activation restrains NUMBL expression and that NUMBL negatively regulates NF-κB activation most likely by targeting TRAF6 and NEMO, both essential for NF-κB activation. To further interrogate this potential mechanism, we treated GFP-and NUMBL-expressing WT bone marrow macrophages with RANKL for 4 days followed by Western blot for TRAF6 and NEMO. We find that NUMBL overexpression results in significant decrease in TRAF6 and NEMO expression (Fig. 5A). As endogenous expression of TRAF6, NEMO and TAK1 is not sufficient to carry out co-immmunoprecipitation and ubiquitination studies since increased NUMBL expression (in TAK1-null cells) will degrade the TRAF6 and NEMO, we employed transfection studies to confirm the NUMBL-mediated degradation of TRAF6, NEMO and TAK1 in Plat-E cells (modified HEK293 cells). To this end, Plat-E cells were co-transfected with increasing amount of TAK1, TRAF6, and NEMO with constant amount of NUMBL or with constant amount of TAK1, TRAF6, and NEMO in the presence of increasing amount of NUMBL. Two day post transfection the cells were lysed and subjected to Western blotting. The results indicate that increasing concentrations of either TAK1, TRAF6 and NEMO do not affect NUMBL expression, but increasing NUMBL concentration results in decrease of TAK1 (Fig. 5B), TRAF6 (Fig. 5C), and NEMO (Fig. 5D) expression. This led us to speculate that NUMBL directly interacts with TRAF6 and NEMO and promotes their K48-pUB mediated proteasomal degradation. To verify this possibility, NUMBL-TRAF6 and NUMBL-NEMO co-transfected Plat-E cells were lysed and immunoprecipitated for TRAF6 and NEMO followed by immunoblotting for K48-pUB and NUMBL. For control, Plat-E cells were individually transfected with NUMBL, TRAF6 and NEMO and immunoprecipitated for TRAF6 and NEMO. Co-immunoprecipitation first shows that NUMBL interacts with TRAF6 (Fig. 5E) and NEMO (Fig. 5F). Abundance of K48-pUB in TRAF6, NEMO and NUMBL transfected cells when compared with cells co-transfected with TRAF6, NEMO combination only shows that NUMBL induces K48-pUB mediated proteasomal degradation of TRAF6 (Fig. 5E) and NEMO (Fig. 5F). The observation that TAK1 overexpression does not affect NUMBL levels (Fig. 5B, left panel), suggests that additional mechanisms regulate baseline NUMBL expression. In this regard, TAK1-mediated degradation of NUMBL may require other interacting partners, which were not over-expressed in Plat-E cells but otherwise have normal expression in primary cells.   Immuno-blot from total cell lysates and TRAF6-immunoprecipitates shows that NUMBL interacts with TRAF6 and leads to its K48-poly-ubiquitine mediated proteasomal degradation. (F) Plat-E cells were co-transfected with NUMBL and NEMO. Immuno-blot from total cell lysates and NEMO-immunoprecipitates shows that NUMBL interacts with TRAF6 and leads to its K48-poly-ubiquitine mediated proteasomal degradation. Full-length Western blots images are included in supplementary data as Figure S5.A-F.

Discussion
NF-κB signaling is crucial to maintain cellular homeostasis in all cell types including OCs and their myeloid precursors. During differentiation of myeloid progenitors into OCs, RANKL triggers formation of a signaling complex encompassing TAK1, TRAF6 and other adaptor proteins that attach to distal motifs of RANK culminating with stimulation of NF-κB signaling. This process eventually leads to induction of transcriptional machinery for OC formation that contains several activators and repressors.
Various regulators of NF-κB signaling have been identified in OCs and other cells, which interact with key players of NF-κB signaling to maintain cellular homeostasis. For example, A20 27 , IRF8 28 , Bcl6 29 , RBPJ 5,7,30,31 have been reported to repress NF-κB signaling during OC differentiation. Other regulators such as PARPs, were shown to upregulate NF-κB activation in OC and other cells types [32][33][34] . Hence, better understanding of molecules and systems that regulate RANKL-induced NF-κB signaling and osteoclastogenesis will offer new insights to regulate this pathway and maintain skeletal health.
In a previous study, we discovered that NUMBL, which is important for determination of cell fates during development, is involved in regulating the osteoclastogenic machinery. Specifically, under TAK1 deficient conditions, elevated levels of NUMBL inhibit OC differentiation by degrading Notch intracellular domain (NICD). This event leads to accumulation of the transcription factor RBPJ, resulting in transcriptional repression of NFATc1 7 . In this current study, we show that NUMBL directly represses NF-κB activation during RANKL-induced OC differentiation by regulating proximal components of NF-κB signaling.
We first observed that increased NUMBL expression in TAK1 null cells coincides with decreased TRAF6 and NEMO expression, both of which are key components of RANKL mediated NF-κB signaling. Moreover, exogenous expression of catalytically active TAK1 not only decreases NUMBL 7 expression but also restores TRAF6 and NEMO expression in TAK1 null cells, suggesting that TAK1, TRAF6 and NEMO may interact with NUMBL and reciprocally regulate OC differentiation, where NUMBL might be acting as a negative regulator of NF-κB activation. NUMBL overexpression studies in BMMs clearly suggest that NUMBL inhibits OC differentiation and function via inhibiting NF-κB activity and NFATc1 expression. Conversely, NUMBL deficient cells exhibit increased OC differentiation and expression of genes associated with OC differentiation. These experiments establish NUMBL as a negative regulator of OC differentiation and function.
NUMBL has been shown to interact with TAB2 (TAK1 binding protein 2) and mediates TRAF6 degradation, thus inhibiting NF-κB activation 24,25 . In glioma cells NUMBL has been reported as a suppressor of TRAF5 mediated NF-κB activation 25 . Based on this and our data, we hypothesized that RANKL-mediated NUMBL regulation during OC differentiation is TAK1-dependent. Gene expression data suggest that in TAK1 sufficient WT cells, RANKL inhibits NUMBL transcription during early stages of RANKL stimulation. At the same time detection of K48-linked ubiquitination in immuno-precipitates using NUMBL antibody in WT and TAK1-null cells shows that, whereas in WT cells TAK1 leads to K48-linked pUB mediated degradation of NUMBL, in TAK1-null cells NUMBL accumulates due to hypo-K48-ubituitiantion and, as a result, reduced degradation. As we have suggested previosuly, NUMBL expression is TAK1-dependent and exerts a baseline osteoclast regulation when expressed. Hence it is possible that RANKL exerts a temporal effect, wherein at early stages of RANKL stimulation TAK1 is activated leading to reduced NUMBL expression and initiation of osteoclastogenesis. As this osteoclastogenesis process is established, TAK1 activity is reduced by feedback mechanisms which coincide with increased expression of osteoclast negative feedback mechanisms, i.e. increased NUMBL expression. Further studies are required to establish the mechanism by which RANKL treatment decreases NUMBL expression at the transcriptional level and TAK1 kinase activity mediated-NUMBL degradation post translationally. It is likely that RANKL-induced TAK1 forms a complex with TAB2 and TRAF6, a well-known E3 ligase, or other yet to be identified ligases to induce ubiquitination of NUMBL. We further speculate that TAK1 may target the inducible expression of NUMBL which is presumably post-transnationally modified and marked for ubiquitination mediated degradation as opposed to the basal levels of un-modified NUMBL.The molecular details of such mechanism await further studies.
To further understand the mechanism of NUMBL-mediated NF-κB regulation, we analyzed TRAF6 and NEMO expression in WT BMMs cells over-expressing NUMBL. Immunoblots suggest that increased NUMBL expression in WT cells is associated with decreased TRAF6 and NEMO expression. As described earlier, NUMBL acts as an E3 ligase and mediates degradation of proteins like TRAF6 24 . Based on this, our overexpression study in Plat-E cells shows that increasing expression of NUMBL leads to decreased expression of TAK1, TRAF6 and NEMO. However, increasing expression of TAK1, TRAF6 or NEMO do not drive down the expression of NUMBL below its normal baseline level. One possible explanation is that TAK1 interaction with NUMBL requires other proteins such as TAB2 and ligases. Given the fact that TAB2 is also an obligatory TAK1 partner, it is possible that TAB2 is a rate-limiting protein that determines the overall homeostatic balance between TAK1 and NUMBL function under RANKL-induced conditions, e.g. NF-κB activation and OC differentiation.
The evidence that NUMBL physically interacts with both TRAF6 and NEMO, and induces their K48-ubiquitination mediated proteasomal degradation further supports its fundamental role as potential RANKL-induced NF-κB signaling repressor. This process is pronounced in the absence of TAK1, suggesting that NUMBL is an innate mechanism that dampens NF-κB activation and osteoclastogenesis at times when activation of TAK1 by RANKL subsides. Using the same rational, it is also possible that NUMBL plays a role in regulating other signaling complex featuring TRAF6 or TAK1 that are required for normal osteoclastogenesis. For example, NUMBL is predicted to interact with atypical-PKCs (a-PKC), which have been shown to form a complex which includes p62/TRAF6/a-PKC upon RANKL stimulation in BMMs 35 .
Since NUMBL expression is normally decreased or low under normal OC differentiation, it will be interesting to determine the expression and regulation of NUMBL in other pathological models of osteolysis such as arthritis. One can speculate that expression level of NUMBL remain at baseline during inflammatory conditions owing to constant activity of TAK1 and elevated expression and ligase activity of TRAF6. Therefore future studies are required to further elucidate the possible role of NUMBL under normal and pathological OC differentiation and explore the utility of this innate mechanism in osteolytic conditions. Collectively, our data present NUMBL as a negative regulator of osteoclastogenesis. Increased NUMBL expression, in one hand, regulates NFATc1 expression via accumulating RBPJ (NICD/RBPJ axis). In the other hand, it directly induces K48-mediated proteasomal degradation of TRAF6 and NEMO to dampen NF-κB activation and attenuate osteoclast differentiation.

Methods
Animals. TAK1 floxed mice on a C57BL/6 background were crossed with LysM-Cre (Jacksons Laboratories) to produce heterozygous mice. The TAK1 heterozygous mice were further intercrossed to generate homozygous null mice (TAK1-cKO). NUMB floxed and NUMBL floxed mice were obtained from Jacksons Laboratories which were bred together to generate heterozygous mice followed by intercrossing to generate homozygous double floxed mice (NUMB/NUMBL floxed termed N/NL floxed). Later the double floxed mice were crossed with LysM-Cre to finally generate a NUMB/NUMBL double knockout (N/NL-dcKO mice). RelA_luc reporter mice were purchased from Taconic Biosciences. Mice were housed at the Washington University School of Medicine barrier facility. All experimental protocols were carried out in accordance with the ethical guidelines approved by the Washington University School of Medicine Institutional Animal Care and Use Committee.
Cell Culture. Bone marrow cells were cultured in α-MEM supplemented with 100 units/ml penicillin/ streptomycin and 10% FBS (v/v) with 10 ng/ml M-CSF for 16 h to separate adherent cells from non-adherent cells. Non-adherent cells were collected and further cultured with M-CSF (20 ng/ml) for 3 to 4 days, followed by RANKL (50 ng/ml) treatment for different time points (as described in the figures). In certain conditions, TAK1-cKO cells were either cultured with TNFα neutralizing antibody (0.2 μg/ml). To understand the interaction of NUMBL with TRAF6 and NEMO and to generate retroviral vectors Plat-E cells were cultured in DMEM supplemented with 100 units/ml penicillin/streptomycin and 10% FBS (v/v) 7 .
Transfection and retroviral infection. For expression studies, various genes like NUMBL, TAK1-WT, TAK1-K63W, TRAF6 and NEMO were cloned in retroviral pMX-constructs. For exogenous expression, relevant pMX-constructs were transfected in Plat-E cells using xtrem gene9 (Roche,). Similarly, for retroviral production relevant pMX-constructs were first transfected into Plat-E cells using xtreme gene 9, followed by collection of virus containing media for 2 days. This virus containing media were later used to infect bone marrow cells 7 .
Actin ring staining. Osteoclasts were generated on bone slices from bone marrow macrophages (BMMs) expressing GFP and NUMBL. For actin ring staining, the cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100, rinsed in PBS, and immunostained with AlexaFluor 488-phalloidin (Invitrogen).
RelA-Luc reporter assay. BMMs isolated form RelA_luc reporter mice (Taconic), were transduced with pMX-GFP and pMX-NUMBL retroviral particles. One day after transduction the cells were cultured in 96 well plate in the presence of M-CSF for two days, followed by starvation and RANKL treatment for 0, 15, 30 and 60 min. Post RANKL transfection cells were lysed and luciferase activity was measured using luciferase assay system (Promega). The luciferase activity was normalized with protein concentration (BCA assay, OD at 592 nm).

Western blot Analysis.
Total cell lysates were prepared in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) from either Plat-E cells expressing different genes or Bone marrow cells induced with RANKL and/or TNFα for different time points as described in the figures). Protein concentration was determined and equal amount of protein was loaded onto SDS-PAGE. After transfer, and blocking in 5% BSA for 1 h at room temperature, membranes were probed with specific primary antibody primary antibodies in 5% BSA in PBS-Tween (1% v/v) for overnight and then washed three times with PBS-Tween (PBST) and probed with secondary antibodies from LI-COR (Odyssey Imager; donkey anti-rabbit/IRDye 800CW/anti-goat/IRDye 800CW anti-mouse IRDye 680RD) for 1 h at room temperature. Membranes were then washed three times with PBST and scanned by using LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE, USA). The NEMO, TRAF6, NUMBL, NFATc1 and TAK1 antibodies were purchased from Santa Cruz, Dallas, TX, USA; K48-Ub antibody was purchased from Cell Signaling Technology, Danvers, MA, USA; β-actin was purchased from Sigma, St Louis, MO, USA.


extfieldsofstudy: Biology Medicine

text:
Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

difficult to reach a single consensus as to a Class I signature, leading to the proposition for further subdivision of the class [14][15][16] . Considering phylogenetic analyses of sequences from a wide range of fungi, bootstrap values support a separation of Class I ascomycota and basidiomycota sequences but are too low to confidently support further subdivisions 17,18 . It is also important to note that while the number of predicted sequences increases, both the forces driving hydrophobin gene evolution and the roles of individual genes remain largely unclear 18 .
While hydrophobins from both phyla have been characterized, high-resolution structures exist only for those of ascomycota origin: the Class I EAS, DewA, MPG1, and Class II NC2, HFBI, and HFBII 8,[19][20][21][22][23] . To address whether basidiomycota hydrophobins share structural and functional features with these proteins, we preliminarily characterized SC16 (hyd1) from Schizophyllum commune and determined its structure. SC16 adopts a distinct structural topology reminiscent of both Class I and Class II hydrophobins. Bioinformatics analyses of predicted hydrophobin protein sequences from 215 fungal species reveal that SC16 is representative of a high identity basidiomycota subdivision of Class I (Class IB). This finding was supported by the NMR-based analysis of the Class IB P. carnosa hydrophobin PcaHyd1.

Results and Discussion
SC16 demonstrates Class I properties. The S. commune hydrophobin SC3 is one of the best functionally characterized basidiomycota Class I hydrophobins to date. However, its ability to self-assemble at 100 μ g/ml without significant aeration 12 precluded its detailed structural characterization. Sequence analysis of the S. commune hydrophobins identified SC16 as sharing 56% sequence identity with SC3 from its first to last cysteine residues ( Figure S1) and preliminary NMR studies confirmed the presence of SC16 monomers in solution at protein concentrations required for structural studies. As such, we hypothesized that while the assembly kinetics of SC16 differed from those of SC3, given their sequence identity, SC16 would reveal functional properties largely consistent with SC3, while offering properties amenable to our characterization approach.
The solution properties of recombinant SC16 produced in Escherichia coli were consistent with those of other Class I hydrophobins 10 . Specifically, upon introduction of an air-water interface by gentle head over tail shaking (i.e. aeration) specific binding to the amyloid specific dye ThT was induced as indicated by the linear relationship between enhanced fluorescence of the amyloidogenic dye and protein concentration (Fig. 1A), while the fluorescence emission maxima remained constant at 483 nm regardless of protein concentration ( Figure S2). Furthermore, after aeration atomic force microscopy (AFM) revealed the presence of rodlet-shaped assemblies on a dried down droplet of SC16 in water on highly oriented pyrolytic graphite (HOPG) (Fig. 1B). No assemblies were observed by AFM in the absence of aeration prior to drying on HOPG at the deposited concentration tested. The ~ 2 nm height of the rodlet assemblies is similar to that reported for SC3 (2.5-3 nm) 24 . The distribution of rodlet widths is comparatively narrower than the distribution of lengths and is centred at approximately 5 nm (Fig. 1C,D). The length distribution is centred at approximately 15 nm. SC16 adopts a unique hydrophobin structure. Detailed structural characterization of SC16 was performed by NMR spectroscopy. Multi-dimensional, heteronuclear spectra of SC16 were of high quality with well-dispersed resonances allowing for the assignment of 84%, 80% and 100% of backbone, side chain and aromatic 1 H, 15 N, and 13 C resonances, respectively ( Figure S3A). Due to chemical exchange and intrinsic disorder, a subset of resonances corresponding to Thr18-Asn25 were not assignable while resonances from residues Gly26-Leu116 were nearly completely assigned. An ensemble of twenty SC16 structures was determined using automated assignment with ARIA and displayed good overall structural statistics and low pairwise root mean  Table S1). SC16 comprised four β -strands (β 1 : Val38-Asp46 β 2 : Leu71-Leu79 β 3 : Gln92-Tyr100 β 4 : Val105-Pro111) and an α -helix (Lys52-Leu60; Fig. 2B). β 1 and β 2 formed a short antiparallel β -sheet linked by an ordered loop (L 1 , also referred to as the Cys3-4 loop) that contained the α -helix. β 3 and β 4 formed a second antiparallel β -sheet connected by a β -hairpin motif (L 3 , Cys7-8). A disordered loop (L 2 ) connected β 2 to β 3 . For our discussion of hydrophobin structure, L 1 , L 2 , and L 3 are considered to be the intervening sequences between the four core β -strands, regardless of the specific structures they adopt. The four β -strands assembled into a β -barrel with a hydrophobic core. A secondary buried hydrophobic region in SC16 existed between the surface of the β -barrel and a hydrophobic face of the α -helix present in L 1 . SC16 adopted an ordered, compact globular structure (17 Å × 17 Å × 35 Å) with the exception of the N-terminal region comprising Lys10-Ser32, L 2 , and Ile112-Leu116 at the C-terminus, which { 1 H}-15 N heteronuclear NOE measurements indicated to be dynamic on the ps-ns timescale ( Figure S3B). The smaller dimensions of the SC16 monomer (17 Å) are similar to the AFM measured height of SC16 rodlets (~20 Å). The eight cysteine residues of SC16 formed disulphide bonds in a pattern consistent with that observed in other hydrophobins. Two disulphide bonds were located within the β -barrel of SC16 and covalently linked β 1 to β 2 (Cys41-Cys76) and β 3 to β 4 (Cys96-Cys109). The two remaining disulphide bonds connected the N-terminal tail to β 3 (Cys33-Cys95) and β 1 to L 2 (Cys40-Cys89).
Using the POPS (Parameter OPtimized Surfaces) algorithm 25 on the core region of SC16 (Cys33-Leu116), we determined that 53% of the solvent accessible surface area of SC16 was hydrophobic. Analysis of the surface charge distribution of SC16 revealed several acidic, basic, and uncharged patches ( Figure S4) but the latter, in particular, was not as prominent for SC16 as for EAS, DewA and MPG1 8,19,20 . Closer inspection revealed this surface to comprise several backbone amide groups and side chain hydroxyl groups but lack hydrophobic side chain groups. This observation is unusual as other Class I hydrophobins typically display large exposed hydrophobic regions, which have been hypothesized to be responsible for mediating self-assembly at hydrophobic interfaces 8,19,20 .
Of the structural features displayed by SC16, some were consistent with those previously observed in hydrophobins while others were unique. SC16 maintained the key features of the hydrophobin fold: a central four-strand β -sheet and four disulphide bonds ( Fig. 2) 8,[19][20][21][22][23] . Notably, in solution its sheet adopted a compact β -barrel structure more similar to the core structures of the Class II hydrophobins HFBI and HFBII (backbone atom root mean squared deviation of 3.0 Å and 2.2 Å, respectively; Fig. 3E) than to those of other Class I and II hydrophobins. These, in contrast to SC16, were observed as either open or irregular barrel structures ( Fig. 3A-D).
Structural features shared between SC16 and the Class II hydrophobins HFBI, HFBII, and NC2, were particularly notable. Specifically, all four proteins consist of a β -barrel with an associated α -helix. However, the location of the α -helix differs in the two proteins. In SC16 it is located within L 1 while for the Class II hydrophobins it resides in L 2 . This difference results in the α -helix being situated on opposite sides of the β -barrel when SC16 is compared to the Class II hydrophobins. Furthermore, the α -helices of the Class II hydrophobins are covalently linked to the core β -barrel through disulphide linkages, while in SC16 a hydrophobic interface is formed between the α -helix and β -barrel. This lack of a disulphide bond in SC16 may allow a greater degree of conformational flexibility to adapt to a structure amenable to rodlet formation; a posit consistent with the observation that SC16 is an active Class I hydrophobin forming rodlets under conditions of aeration. Comparing the secondary structural elements surrounding the β -sheet core of the structurally characterized hydrophobins, it becomes apparent that the regions connecting the β -sheet strands (L 1 -L 3 ) are variable in sequence, length, structure, and dynamics amongst members of the protein family (Fig. 3). In SC16, these connecting loops are most structurally similar to those of the Class II hydrophobins. As described above, in Class II, L 2 contains an α -helix while L 1 and L 3 are very short. In contrast, the corresponding regions in the Class I hydrophobins (EAS, DewA, and MPG1) are much more structurally diverse and the extent of dynamics within the loop regions vary. In EAS, L 1 and L 3 are large and unstructured and L 2 contains a β -strand. In MPG1 these long loops contain α -helical regions and in DewA they include both α -helices and β -strands (Fig. 3). Considering these regions within SC16, L 1 and L 3 are ordered while they are flexible in EAS 8 . In DewA all three loops appear well ordered 19 , while in MPG1, only L 1 displays conformational flexibility 20 . The L 2 region is dynamic only in SC16.
The NMR-derived secondary structure of the hydrophobin RodA 26 has been recently reported and it has some similarities with the structure of SC16. In both RodA and SC16 a four-strand β -sheet is observed, and an α -helix is present in L 1 while L 2 appears to be largely disordered. There are potential structural differences between these proteins as well since L 3 is predicted to be disordered in RodA while in SC16 it is incorporated into the β -strands. Finally, an α -helix is predicted to be present in the N-terminal region of RodA that is not found in SC16.
Rodlet formation mechanism of SC16. Hydrophobin rodlet formation mechanisms have been refined based on the structures of EAS 8,27 , DewA 19 , and MPG1 20 . In recent models, the L 3 loop is proposed to undergo a structural transition to a β -strand that oligomerizes with the L 3 of other monomers to form amyloid rodlets 27 . While the dimensions of the SC16 rodlets measured by AFM are consistent with this model, the L 3 of SC16 is a β -turn that connects β 3 and β 4 , and is thus unavailable for amyloidogenesis. Only the L 1 and L 2 regions of SC16 are long enough to potentially undergo the conformational changes required for rodlet formation. Interestingly, the Waltz algorithm 28 predicts that residues 40-47 of L 1 in SC16 are amyloidogenic. Although our data indicate this loop is not dynamic and is folded into an α -helix, it may still possibly undergo structural rearrangement. Similar structural rearrangements have been observed for the closely related hydrophobin SC3, which forms a transient α -helical structure before undergoing further structural transitions to form a β -sheet rich rodlet 29 . Currently, it is not known whether a single loop region is responsible for amyloidogenesis in all known structures. Although this phenomenon has been linked to L 3 in EAS, L 3 is not likely to initiate such an assembly in SC16 or DewA, while in MPG1 the role of L 3 is unclear. In contrast to SC3 12 , SC16 only forms rodlets upon aeration. This finding is similar to MPG1, where agitation is necessary for rodlet assembly as it occurs via a surface driven mechanism. This discrepancy could simply be due to variation of protein concentrations and buffer conditions in our studies since rodlet assembly of SC3 is both concentration and buffer dependent 12 . Intriguingly, L 1 of SC16 buries ~100 Å 2 of hydrophobic surface area that could become accessible if L 1 were to undergo a structural rearrangement when positioned at an interface, allowing for amyloidogenesis. Finally, glycosylation of SC3 was demonstrated to contribute to the kinetics of assembly, stability of solution monomer, and stability of films 30 . Based on primary sequence, SC16 is predicted to have fewer glycosylation sites 31 . These insights suggest that minor sequence differences or glycosylation status may be responsible for fine-tuning the functions of hydrophobins.

SC16 is a representative member of a Basidiomycota Class I hydrophobin subdivision (Class IB).
To contextualize the structural and functional properties of SC16, we constructed a database of confirmed and predicted hydrophobin sequences from the genomes and transcriptomes of 215 unique filamentous fungi (72% Basidiomycota, 28% Ascomycota). The bias towards the Basidiomycota phylum is a reflection of the available genomic and transcriptomic data as Ascomycota species are comparatively understudied 32 . A total of 1046 canonical sequences were identified as predicted or confirmed hydrophobins. Using the PFam database of protein families 33 , 781 sequences were assigned as Class I (PFam01185), and 215 to Class II (PFam06766), while 50 were not predicted to belong to the hydrophobin family, including the well-characterized Class I EAS. PFam classifications of other previously biophysically characterized hydrophobins are consistent with their experimentally determined classes 10 .
To more comprehensively assess the differences and similarities between hydrophobin sequences, we conducted a principal component analysis (PCA) of the sequence alignment matrix of the canonical set (Fig. 4A,B). Briefly, sequences that cluster together along a principal component tend to share similarities, the degree of similarity increasing with distance from the origin. The first principal component (PC1) describes the most significant differences in the data while the differences described by PC2 are less significant in the context of the total data, but could prove to be more significant for a subset of sequences.
In this analysis, both phylum and PFam prediction of hydrophobin class correlate with position in the PC2 vs. PC1 plot (Fig. 4A,B). Class II hydrophobins are readily distinguished as they form a distinct cluster, a result consistent with the established high sequence conservation of the class 17 . In contrast, class I hydrophobins reside in all four quadrants of the plot displaying, as expected, comparatively high sequence variability 7 . In PC2, however, there is a distinct separation of Ascomycota and Basidiomycota Class I sequences, with an intermediate region containing sequences originating from both phyla consistent with previous phylogenies 17,18,34 . The mixed-region of Class I contains all previously structurally characterized Class I hydrophobins and has little sequence or loop length conservation, an observation consistent with the current description of the Class I group of hydrophobins in the literature 11 .
The furthest clusters from the origin (i.e., twice as far as any other in PC2) are Class I Ascomycota (Class IA) sequences that include the Aspergillus fumigatus RodA. This suggests these sequences diverge from those of other hydrophobins in high identity clusters, a result consistent with previous phylogenies 18 . However, what is particularly unique about this analysis is the emergence of a highly conserved Class I subdivision consisting of Basidiomycota sequences (Class IB). Furthermore, this subdivision includes the experimentally confirmed Class I sequences HGFI (Grifola frondosa), VMH2 (Pleurotus ostreatus), along with the S. commune SC3 and SC16.
To further appreciate the sequence-based clustering of Class I hydrophobins, we generated alignments of the sequences located within 14 equal-sized regions across PC2 vs. PC1 ( Fig. 4C; full alignments in Figure S6A-AB, tables of identities in Table S2A-L). Consistent with the clustering of Class IB in the PCA, the 6 consensus sequences from Class IB hydrophobins contain similar loop lengths and a number of conserved residues in addition to the cysteine pattern throughout the hydrophobin sequence. The Class IA and Class II hydrophobins were also observed to have similar loop lengths and additional conserved residues within their groups. In contrast, very little sequence conservation is observed for the mixed region. This suggests that hydrophobins within the Class IA and IB regions may have similar structural and functional properties to other members of their respective groups.
To validate this hypothesis, we collected structural data on another predicted Class IB hydrophobin PcaHyd1 (Phanerochaete carnosa), which is located at the opposite side of the Class IB sequence cluster from SC16. Using NMR spectroscopy, we completed the backbone 1 H, 13 C, and 15 N chemical shift assignments for PcaHyd1 and calculated the chemical shift index for each residue of the protein 35 . The predicted secondary structure elements of PcaHyd1 aligned with the structured regions of SC16 ( Figure S5, which is consistent with the high sequence conservation within the core regions of SC16 and PcaHyd1 (42% and 59% sequence identity and similarity, respectively). Overall, it is likely that SC16 and PcaHyd1 share the same three-dimensional fold, which would be common to Class I Basidiomycota hydrophobins given the high sequence similarity observed for this Class.
All previously determined Class I hydrophobin structures (i.e., EAS 8 , DewA 19 , and MPG1 20 ) display distinct structural features from each other and cluster in the mixed region of the PCA analysis, which contains large sequence variations. This suggests that these structures are not broadly applicable to Class I sequences. In contrast, SC16 is representative of the Class IB hydrophobins, given the high sequence identity of the subdivision and similar structures of SC16 and PcaHyd1. Such an integrated structure-bioinformatic analysis will lead the way to characterizing the sequence determinants of hydrophobin function and improve our understanding of functional amyloidosis.

Materials and Methods
Bioinformatic analysis of hydrophobin sequences. The UniProt, JGI, and NCBI databases were mined for the canonical eight cysteine pattern residue: > = Cys to the left of the first CC, > = Cys between the two Cys, and > = Cys to the right of the second CC (see SM for more details). The resulting FASTA sequences were submitted to signal IP using default parameters to determine the predicted cleavage site of the secretion signal and to PFam 33 for domain assignation. Sequence alignment of the resulting 1046 mature predicted and confirmed hydrophobins was constructed using MAFFT (multiple alignment using fast fourier transform) 36 . Using this sequence alignment, Jalview (vs 2.8.2) 37 was used to produce a principal component analysis of the sequence alignment matrix using default parameters.
Plasmid construction, protein expression and purification. DNA coding for residues Thr18-Leu116 of the Hyd1 (SC16) gene of Schizophyllum commune or residues Thr21-Leu138 of the Hyd1 gene of Phanerochaete carnosa were cloned into a pET32 vector downstream of DNA coding for His 6 -tagged thioredoxin (Trx) and an enterokinase cleavage site. The N-terminal signal peptide was not cloned and after cleavage the amino acids KAMADIGS remain at the N-terminus of this SC16 construct. Cultures of Origami B (DE3) E. coli were grown in LB media supplemented with 15 μ g/mL kanamycin, 12.5 μ g/mL tetracycline, and 100 μ g/mL carbenicillin at 37 °C until the culture reached an O.D. 600 of 0.6-0.8. Protein expression was induced with 300 μ M isopropyl β -D-thiogalactopyranoside and occurred for 20 hours at 23 °C.
After expression, cell pellets containing Trx-SC16 were lysed in lysis buffer (20 mM Tris-HCl pH 8, 250 mM NaCl), clarified by centrifugation and the Trx-SC16 protein was isolated via Ni 2+ affinity. His 6 -tagged Enterokinase was added in a ratio of 1 unit per 500 μ g of protein to remove the Trx tag, and the sample was dialyzed against 20 mM Tris-HCl pH 8, 150 mM NaCl, and 2 mM CaCl 2 . After protease cleavage, the Trx and enterokinase were separated from SC16 by a second round of Ni 2+ affinity chromatography and then concentrated using ultrafiltration. Purified protein was either used immediately for experiments or lyophilized in 20 mM ammonium acetate at pH 4.5 and stored in a desiccator for long-term storage. The observed molecular weight of purified SC16 corresponded to its calculated molecular weight of 10.8 kDa. The PcaHyd1 protein was prepared using the same procedure as SC16. For NMR sample preparation the E. coli culture was grown in M9 minimal medium supplemented with 1 g/L 15 NH 4 Cl, 2 g/L 13 C-glucose, and 10 mL/L of 13 C/ 15 N-BioExpress-1000 medium (Cambridge Isotope Laboratories).
Thioflavin T binding assay. SC16 was resuspended in 20 mM phosphate buffer (pH 7.5) to a concentration of 66 μ g/ml. The solution was left to incubate at ambient temperature for 12 hours (referred to as "overnight shaking") either with or without gentle mixing head over tail shaking on a rotary shaker at approximately 40 rotations per minute. After incubation, Thioflavin T was added to each solution to a concentration of 4 μ M. The protein samples were then diluted as specified and emission spectra were collected (scanning between 450 and 600 nm, with 5 nm slit widths) using an excitation wavelength of 442 nm with 2 nm slit widths (Cary Eclipse Fluorescence Spectrophotometer, Varian).
This solution was used to prepare a 1 ml solution at 2 μ g/ml in a microfuge tube with 1 ml empty headspace. This solution was agitated using vortexer set on high for 30 minutes. A 2 μ l sample from the core of the solution was then placed directly on freshly cleaved HOPG and left on the bench to dry for 30 minutes. Samples were placed in a desiccator overnight before imaging.
All atomic force microscopy (AFM) images were acquired using a Nanoscope IIIA Multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA). The AFM images were collected using Nanoscope version 5.12r3 software and a J scanner that has a maximal lateral scanning area of 125 μ m × 125 μ m. Silicon probes TESP-V2 (Bruker AFM Probes, Camarillo, CA) were irradiated under UV light for 30 min to remove possible organic contaminants. All images were captured as 512 × 512 pixel data sets at a tip scan rate between 0.9 and 1.1 Hz with a cantilever drive frequency of between 280 and 320 kHz.
The length and width values of SC16 rodlets were determined in ImageJ (version 1.46r) 38,39 using the following sequence of image processing steps: (1) unsharp filter with radius of 2 pixel and mask weight of 0.6; (2) despeckle; (3) binary threshold; (4) particle analysis using fit ellipse measurement with size from 0 to infinity and circularity from 0-0.7. The height values were determined by analyzing cross sections of the rodlets using NanoScope Analysis software version 1.50. NMR spectroscopy and structure calculations. NMR spectra were recorded on a Varian 600 MHz INOVA spectrometer equipped with a triple-resonance probe at 30 °C. A single protein sample containing 1 mM 13 C/ 15 N-labeled SC16 in 25 mM Tris-HCl pH 8.0 and 150 mM NaCl was used for data collection. Standard triple resonance experiments were used to assign the backbone and side-chain resonances of SC16. Distance restraints were derived from 15 N edited NOESY-HSQC and both aliphatic and aromatic 13 C edited NOESY-HSQC spectra collected with a 100 ms mixing time. In order to identify residues involved in hydrogen bonds, a 1 H-15 N HSQC spectrum was collected on a D 2 O exchanged sample of SC16. Spectra were processed using NMRPipe 40 and analyzed using CcpNmr Analysis version 2.4 40 . Data was collected for PcaHyd1 under the same sample conditions as SC16, except that data collection occurred at 25 °C.
Backbone and side-chain chemical shift values, partially assigned NOESY peak lists and dihederal angle restraints generated by DANGLE 42 were used as inputs to ARIA version 2.3 43 for automated NOE assignment and structure calculation of SC16. From the initial structural folds, it was clear that SC16 contains the conserved disulphide bonding pattern of hydrophobins and these four restraints were added to the structure calculation. Hydrogen bond restraints were added for residues that have strong signal in a 1 H-15 N HSQC spectrum for a D 2 O exchanged sample as well as for the helical regions of the initial ensembles. The final structural ensemble of SC16 consists of the 20 lowest energy structures out of 200. These structures were refined in water by CNS 1.21 44,45 . The quality of the final ensemble of structures was assessed through RPF analysis 46 , PROCHECK 47 and the PSVS suite 48 . The PyMOL Molecular Graphics System (Version 1.7.1 Schrödinger, LLC) was used for visualization, distance measurements, and figure generation. Chemical shifts and the final structural ensemble of SC16 have been deposited into the Protein Data Bank and the BioMagResBank (Accession numbers 2NBH and 25976, respectively). The chemical shift assignments of PcaHyd1 were deposited into the BioMagResBank (Accession #: 26907).