Mucor and nine Penicillium strains) isolated from dairy products, then investigate the susceptibility of the molds toward 12 LAB cultures. Fungal cell growth and morphology in malt extract broth was monitored using oCelloScope at 25°C for 24 h. Mucor plumbeus 01180036 was the fastest growing and Penicillium roqueforti ISI4 (P. roqueforti ISI4) the slowest of the tested molds. On yogurt-agar plates, all molds grew at 5, 16, and 25°C in a temperature-dependent manner with Mucor strains growing faster than Penicillium strains regardless of temperature. The sensitivity toward 12 LAB cultures was tested using high-throughput overlay method and here all the molds except P. roqueforti ISI4 were strongly inhibited. The antifungal action of these LAB was confirmed when spotting mold spores on agar plates containing live cells of the LAB strains. However, if cells were removed from the fermentates, the inhibitory effects decreased markedly. The antifungal effects of volatiles tested in a plate-on-plate system without direct contact between mold and LAB culture media were modest. Some LAB binary combinations improved the antifungal activity against the growth of several molds beyond that of single cultures in yogurt serum. The role of competitive exclusion due to manganese depletion was examined as a possible antifungal mechanism for six Penicillium and two Mucor strains. It was shown that this mechanism was a major inhibition factor for the molds tested apart from the non-inhibited P. roqueforti ISI4 since addition of manganese with increasing concentrations of up to 0.1 mM resulted in partly or fully restored mold growth in yogurt. These findings help to understand the parameters influencing the mold spoilage of dairy products and the interactions between the contaminating strains, substrate, and bioprotective LAB cultures.Fungal spoilage of dairy products is a major concern due to food waste and economical losses, some fungal metabolites may furthermore have adverse effects on human health. The use of lactic acid bacteria (LAB) is emerging as a potential clean label alternative to chemical preservatives. Here, our aim was to characterize the growth potential at three storage temperatures  of a panel of molds (four  Penicillium and Mucor genera, are involved in dairy product spoilage  is recognized as an opportunistic pathogen, which can produce the mycotoxin 3-nitropropionic acid  strains  was shown to have growth inhibitory effect on Penicillium aurantioviolaceum (P. aurantioviolaceum) and Mucor plumbeus (M. plumbeus) (Lactobacillus paracasei (L. paracasei) DGCC 2132 displayed antifungal properties against Penicillium solitum (P. solitum) DCS 302 and Penicillium sp. nov. DCS 1541 in yogurt  have commonly been reported as potential biopreservation tools to inhibit fungal growth in different foodstuffs, extend shelf life, and sometimes even improve the nutritional value and sensory characteristics . For exalumbeus)  and Lactn yogurt . The mecn yogurt . Leyva SThe aim of the present study was to: (a) characterize the growth potential at three temperatures of a panel of molds isolated from freshly fermented dairy products such as yogurt or skyr; (b) investigate the susceptibility of the molds toward different preparations of LAB by assessment of the direct interactive effect of molds with the LAB fermentates with or without living cells, as well as no direct contact assessing the role of volatiles; (c) evaluate the inhibitory effects of 12 LAB cultures in selected combinations on the growth of these molds and (d) investigate the role of competitive exclusion due to manganese depletion in inhibition.6 spores/mL and stored at −80°C until further use. Manganese chloride (MnCl2) was obtained from Sigma-Aldrich  and used for the evaluation of manganese depletion. Commercial plain yogurt  was purchased from the local supermarket .The 12 LAB strains used in this study are listed in TM combining optical techniques. One hundred microliters of spore suspension of each mold (1.0 × 103 spores/mL) in MEB medium were added into a 96-well microplate (approximately 100 spores/well). The 96-well microplate was standing at room temperature for around 1 h after inoculation to allow the spores to settle in these wells, and then placed in the oCelloScope system. The samples were measured automatically and the instrument-derived growth values were recorded every 60 min for 24 h. Automated growth kinetic analyses were performed using the Background Corrected Absorption (BCA) algorithm {BCA = log10[Σ(corrected absorption pixel histograms)]} of the oCelloScope-specific software, UniExplorer (v.5.0.3). For each mold strain, growth testing was performed with three technical replicates.Mold cell growth was monitored at 25°C for 24 h as described by 5 spores/mL) on yogurt-agar plates  in triplicate (2.0 × 103 spores/spot)  . Plates 7 CFU/mL) was spotted on the center of each well and the 24-well microplates were incubated at 37°C for 48 h under anaerobic conditions. Thereafter, wells were overlaid with 100 μL of 0.5% malt extract soft agar inoculated with the individual mold at a final concentration of 1.0 × 103 spores/mL (100 spores/well). Wells inoculated with molds but without LAB cultures were used as control. The 24-well microplates were incubated for up to 8 days at 25°C. The antifungal activity was determined by visually evaluating fungal growth as strong inhibition (+++), weak inhibition (++), fungal growth with delayed sporulation (+), and no inhibition (−) on the basis of the inhibition zone . Un-inoculated MRS broth kept at 37°C for 22 h was used as control. The antifungal activity of 12 LAB fermentates with or without live cells against target molds was measured using an agar spot method, and the contribution of volatile compounds produced by LAB to the antifungal activity was assessed in a “Plate-on-Plate” test system  on these LAB fermentates agar plates in triplicate and incubating at 25°C for 5 days. Control groups were prepared by spotting each mold suspension on the plates prepared by mixing un-inoculated MRS broth with MEA. Multispectral images of plates with spotted molds were captured by Videometer Lab2 spectral imaging instrument and the size of each mold colony was calculated based on the number of pixels using MATLAB 2018b software (The agar plates (1% agar) of each LAB fermentate with bacterial cells were prepared by mixing with melted, tempered MEA (in a 48°C water bath). These agar plates were used after solidification and a short drying period. The antifungal activity of each LAB culture was tested by spotting 20 μL of each spore suspension  in triplicate. On top of the MEA plate, a plate with LAB fermentate agar containing live cells was placed upside down and the two plates were sealed together with parafilm. Control plates were prepared by mixing MRS broth with agar. The inhibitory effect of volatiles produced by bacterial cells on mold growth was assessed after 5 days at 25°C as described above.An MEA plate was spotted with 20 μL of each mold spore suspension  and subsequent filtration of the supernatant through a 0.22-μm pore size filter for sterilization . The serum was kept at 4°C prior to use.Yogurt serum, which was used as a transparent yogurt surrogate in this assay, was prepared by centrifugation of the commercial plain yogurt for 30 min at 4, 400 × L. plantarum LP37 was selected as the main culture and combined with each of the other eleven cultures. Yogurt serum was inoculated with the single or combined cultures (in the ratio of 1:1) with the initial inoculum level of 105 CFU/mL, and then fermented at 37°C for 22 h. After fermentation, the agar plates were prepared by mixing the fermentates of single or combined cultures, respectively, with melted, tempered agar. Un-inoculated yogurt serum agar plates (1% agar) were used as control. The inhibitory effect of LAB binary combinations and each LAB single culture was assessed by spotting spore suspension with 20 μL of 1.0 × 105 spores/mL of each mold on these fermentate-agar plates, respectively. The four Mucor strains were incubated at 25°C for 2 days due to their rapid growth while the nine Penicillium strains were incubated for 5 days.The antifungal activity of LAB binary combinations in yogurt serum was tested by pre-fermentation of LAB cultures either alone or in combination . Here, LL. plantarum LP37 was selected as a representative, inhibitive strain for exploring the role of manganese depletion in the growth inhibition of six Penicillium strains and two Mucor strains, representing different species of spoilage strains. The commercial plain yogurt was inoculated with L. plantarum LP37 at a final concentration of 1.0 × 107 CFU/mL, and then distributed into four bottles, which were subsequently supplemented with manganese ion at the final concentration of 0, 0.001, 0.01, and 0.1 mM, respectively  of each mold were spotted on these yogurt agar plates in triplicate. After incubation for 5 days at 25°C, quantification of mold growth was performed as described in Subsection “Sensitivity of the Molds Toward Different Preparations of LAB.”In this test, ectively . Yogurt L. plantarum LP37. In brief, L. plantarum LP37 was inoculated in yogurt serum at a final concentration of 1.0 × 107 CFU/mL and then fermented at 37°C for 22 h prior to use. Both L. plantarum LP37 fermentates with or without bacterial cells were used for testing the manganese depletion. Manganese ion was added into yogurt serum as described above and the agar plates were prepared by mixing with agar. The pH and number of CFU/mL of L. plantarum LP37 were determined before and after fermentation. Un-inoculated yogurt serum kept at 37°C for 22 h was used as control. Each spore suspension was spotted as described above.In addition, manganese depletion test was carried out in yogurt serum as well after pre-fermentation with P < 0.05 . Statistical analyses were performed using SPSS software ver. 26.0 .All the tests in this study were done in triplicate and the values were expressed as mean values ± standard deviation (SD). The statistical significances of experimental antifungal activity of LAB combinations were evaluated by one-way analysis of variance (ANOVA), differences were considered statistically significant at Mucor strains generally grew faster than the Penicillium strains with M. plumbeus 01180036 as the most rapidly growing isolate. M. circinelloides 01180023 was the first isolate to reach stationary phase after approximately 10 h but reached a lower maximum level than the other strains. The fastest growing isolate among Penicillium strains was Penicillium glabrum ISI3, which grew almost as fast as the M. plumbeus strains. In contrast, P. roqueforti ISI4 cells displayed the slowest initial growth with respect to other strains tested. In addition, the morphological changes of molds, such as hyphae development, could also be studied visually from the oCelloScope images and quantified using morphological descriptors  at 5°C, while this took 4 days at 16°C and 3 days at 25°C. At 5°C .At the three tested temperatures, 5, 16, and 25°C, all tested molds were capable of growth on yogurt agar plates  in a tem. At 5°C , mold gr at 16°C , the gro at 25°C  were in 7 CFU/mL except P. roqueforti ISI4, which showed resistance to all the tested LAB cultures except for a weak inhibition when exposed to Lentilactobacillus parabuchneri LPB04  by all the 12 LAB cultures at the concentration of 10ri LPB04 . The spoP. roqueforti ISI4, which showed no or very weak inhibition as also seen previously in the high-throughput overlay test. In comparison with the results of the high-throughput overlay test, more differentiation of the response was observed since some strains  were only inhibited to a lesser degree (up to 50%). Comparing with other LAB cultures, L. plantarum LP48 showed weaker inhibition on P. commune 01180015 and P. palitans PPa01 with inhibitory effect of around 60 and 75%, respectively. The inhibitory effect of L. paracasei LPC46 on M. circinelloides 01180023 was only around 50%. Although some fermentates  displayed a slight inhibitory effect on P. roqueforti ISI4 of around 20–25%, these LAB cultures did not have the capacity to strongly inhibit the growth of this mold. After incubation for 9 days (data not shown), some tested molds were still strongly inhibited by the fermentates, while for other molds, some growth was observed after the 9 days, indicating a more fungistatic rather than fungicidal effect.The antifungal activity of 12 LAB fermentates with live cells in MRS against tested molds was tested. Results indicated that the inhibitory effect of the LAB fermentates with cells varied drastically . In geneP. commune 01180002 being more sensitive, followed by P. solitum ISI5, whereas P. roqueforti ISI4 again was largely unaffected. Among the four LAB species tested, the L. plantarum cell-free fermentates gave rise to the strongest inhibitory effects whereas L. parabuchneri cell-free fermentates lost most of their effect. In addition, the antifungal compounds produced by L. plantarum LP01 displayed stronger inhibition on P. commune strains.In this assay, the sensitivity of molds to LAB cell-free fermentates was determined in MRS medium. As seen in L. parabuchneri cultures here displayed higher inhibitory effect compared with other LAB cultures. The volatiles produced by L. parabuchneri LPB02 showed the highest inhibitory effects on P. commune 01180002 (60%), followed by Penicillium crustosum 01180001 and P. glabrum ISI3. Since the cell-free fermentate of the same strain, L. parabuchneri LPB02, gave rise to a relatively weak inhibition of these molds, this indicated that the activity of L. parabuchneri LPB02 grown in MRS against molds partly relied on volatiles.In order to test the antifungal volatiles, a plate-on-plate system was employed. Here, the inhibitory effect of volatile compounds produced by the 12 LAB cultures was modest, ranging from 0 to around 60% . In contL. plantarum LP37 was selected as a representative for the inhibitory cultures and used in binary combinations with each of the other eleven LAB cultures with an initial approximate inoculum level of 105 CFU/mL. As shown in L. plantarum LP37 combined with either L. rhamnosus LRH14, L. plantarum LP01, or L. plantarum LP48, showed significant improvement of antifungal activity against P. commune strains and/or P. palitans PPa01 compared with the corresponding single cultures (P < 0.05). P. roqueforti ISI4, which was neither inhibited by L. plantarum LP37 nor L. paracasei LPC46 on their own, was weakly inhibited by their combination, indicating some synergistic effect  partly or fully restored the growth of the five sensitive Penicillium and two Mucor strains, suggesting that manganese depletion by L. plantarum LP37 plays a dominating role in the inhibition observed in these strains  rather than the pH 4.6 of yogurt.Different molds can spoil fresh, fermented dairy products but they may have different characteristics affecting their response. Here, the growth potential of n system , during n system . Growth ormation . All strormation . For exaP. roqueforti ISI4. The observed robustness of P. roqueforti toward a number of LAB cultures may be a common characteristics within this species, since this feature has also been observed in other P. roqueforti strains  and L. plantarum exhibited inhibitory effect on Mucor and Penicillium strains.The antifungal activity of 12 LAB cultures against the molds was screened in MRS medium using a high-throughput overlay method in 24-well microplates. The high-throughput antifungal overlay method allows for a rapid screening of a large variety of antifungal agents and the approach is feasible for many laboratories due to the ease and low equipment cost. In this system, all the LAB cultures had strong activity toward all the molds except  strains . Althougrvatives . Using tP. roqueforti ISI4, toward the 12 LAB cultures was confirmed in an agar spot test on fermentates with cells grown in MRS. This test, however, allowed for a slightly more differentiated response. When the cells were removed from the fermentates, the mold growth was still partly suppressed but the antifungal activity decreased markedly in comparison with that of fermentates with live cells. The remaining antifungal activity of cell-free fermentates in MRS is probably due to the presence of various active compounds produced by these LAB cultures in the supernatant such as organic acids, fatty acids, phenolic compounds, proteinaceous compounds and hydrogen peroxide (H2O2) which have been described as being antifungal  under aerobic conditions. L. plantarum LP37 was selected as a representative of an inhibitory LAB and was capable of inhibiting growth of 5 out of 6 selected Penicillium spp. (P. roqueforti was unaffected) and two Mucor strains in yogurt and yogurt serum. In all cases of inhibition, the growth could be partly or fully restored by addition of manganese up to 0.1 mM, thus illustrating that not only the Penicillium but also the Mucor strains were inhibited by the lack of accessible manganese. The inhibition of mold growth in yogurt therefore seems to be associated with the ability of the L. plantarum culture to efficiently scavenge manganese, further supporting that in dairy products the removal of manganese accessible to molds is a main inhibitory mechanism of bioprotective cultures. This also implies that the same cultures may not have the same effect if the products are based on or mixed with materials with high manganese content such as plant materials.Although laboratory media optimized for growth of LAB cultures are well suited for an initial screening of the antifungal activity of the cultures, they do not reflect the composition of the relevant foods nor previous starter culture activity. Further investigations were therefore conducted in yogurt and yogurt serum in order to simulate the conditions in yogurt better. Manganese, an essential trace element, is a key cofactor for the growth of mold  and the L. plantarum LP37 exhibited stronger antifungal activity against both Mucor and Penicillium strains in MRS medium than observed in yogurt or yogurt serum  against some molds, two P. commune strains, compared with single cultures (The enhancement of the inhibitory effect by employment of combinations of different LAB cultures or mixed with propionibacteria has been noted in several studies  and sevecultures . It is nOverall, the interaction between LAB and spoilage fungi is very complex and the LAB will exert antifungal activity in different modes depending on the circumstances making it challenging to develop all-purpose bioprotective cultures. On the other hand, selection of cultures for a particular environment such as plain yogurt or similar dairy products, can be facilitated by recognizing the role of manganese accumulation capacity in this environment. Further work should be undertaken to define which interaction mechanisms are at play in different scenarios and how factors such as oxygen and production of reactive oxygen species, as well as compositional changes of the matrix, influence the effect of the bioprotective cultures.Mucor strains grew faster than Penicillium strains both in MEB medium and yogurt. All tested molds, except P. roqueforti ISI4, were strongly inhibited by the 12 LAB strains when they were grown in MRS. After removal of the LAB living cells from MRS based fermentates, the inhibitory effect decreased, and the effect of volatiles alone was even less, indicating that preformed compounds played a role but they were not the main inhibitory factor. When yogurt serum was employed as a medium, a reduced LAB growth and a weaker inhibition of mold growth were observed, while removal of the cells caused almost complete loss of activity. The inhibition efficacy could in some instances be enhanced by combining cultures. In yogurt, it was shown that manganese depletion by L. plantarum LP37 played an important role in the growth inhibition of both the Penicillium and the Mucor strains tested. Apart from the non-affected P. roqueforti ISI4, it therefore seems that these spoilage molds are generally sensitive to bioprotective cultures capable of making the manganese unavailable in the matrix. Elucidation of the importance of growth conditions, manganese sources, and the rate of manganese transport would further help to expand the use of bioprotective cultures in similar matrices. The inhibitory effect and mode of action of the bioprotective cultures thus seems to depend not only on the target organisms, and growth of the culture but also on the composition of the matrix.Dairy products are very susceptible to spoilage molds, and it is therefore crucial to characterize the growth potential of molds. Here, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CS designed and performed the experiments, contributed to the conception and data analysis, and prepared the manuscript. SK designed and supervised the project, analyzed the data, and reviewed the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, steric constraints in adsorption and variations in S-coordination means that not all S-vacancy sites should be considered equally active. Here, we use a combination of atom-resolved scanning probe microscopy and density functional theory to reveal how the generation of S-vacancies within MoS2 nanoparticles and the subsequent adsorption of thiophene (C4H4S) depends strongly on the location on the edge of MoS2. Thiophene adsorbs directly at open corner vacancy sites, however, we find that its adsorption at S-vacancy sites away from the MoS2 particle corners leads to an activated and concerted displacement of neighboring edge S. This mechanism allows the reactant to self-generate a double CUS site that reduces steric effects in more constrained sites along the edge.The catalytically active site for the removal of S from organosulfur compounds in catalytic hydrodesulfurization has been attributed to a generic site at an S-vacancy on the edge of MoS 2 nanoparticles catalyze the extraction of heteroatom S in hydrocarbons by adsorption onto S vacancies. Here, the authors show that S vacancy properties are highly site sensitive and that adsorption of thiophene leads to self-generation of a more open double vacancy site.MoS Extensive studies involving reaction kinetics, atom-resolved microscopy, X-ray and IR spectroscopy, and density functional theory (DFT) have made it evident that the active sites are located on the edge of single-layer MoS2 catalyst10. The nature of these sites on the catalytically active edges was studied by selectively adsorbing probe molecules such as CO or NO14. These experiments allowed the titration of the number of these sites and its correlation with catalytic activity. Thiophene (C4H4S) has furthermore often been used as an S-containing reactant molecule to probe HDS activity of a catalyst20. This simple five-membered aromatic heterocyclic molecule is relatively inert as its aromatic ring makes its C–S bond quite resistant to rupture. Despite the many fundamental studies devoted to a theory- and experiment-based understanding of the HDS of thiophene a comprehensive mechanistic understanding of this process is still lacking. HDS may proceed by two independent routes: the dominant route for thiophene is the direct desulfurization (DDS) pathway, which involves the direct hydrogenolysis of the C–S bond. The generic type of active site considered for the DDS pathway is a coordinately undersaturated site (CUS) located at a sulfur vacancy (VS) on an edge site formed by reaction with H227. The concentration of the CUS sites has been shown to be dependent on the chemical potential of hydrogen and sulfur in the gas phase and the type of MoS2-edge35. In parallel to DDS, the hydrogenation pathway (HYD) can proceed, in which thiophene adsorption is followed by hydrogenation before C–S bond scission40. The HYD route involves a different type of active site4, proposed to be brim sites located at the top of the MoS2 particle edges8. The HYD route becomes more pronounced compared to DDS for large S-containing molecules such as dibenzothiophene and prevailing for alkyl-substituted derivatives, such as 2,5-dimethyldibenzothiophene42. However, whether the DDS route can still proceed efficiently for such large molecules, which are sterically hindered in their adsorption on a single VS site, remains an open question and a target of intense industrial HDS catalyst development due to the comparatively lower hydrogen consumption in DDS.Catalytic hydrodesulfurization (HDS) is an industrial process applied to remove sulfur heteroatoms from gas oil feeds to reduce sulfur content in fuels and petrochemicals to a <10 ppm level, compatible with legislative specifications. The primary catalysts used for removal of sulfur from crude oil are based on molybdenum disulfide  and DFT on corresponding MoS2 was first experimentally evaluated by atom-resolved STM imaging of single-layer MoS2 nanoparticles. We have specifically characterized S vacancies on hydrogen-activated MoS2-edge structures and then assessed the adsorption of thiophene on these sites by use of a model system composed of well-defined MoS2 nanoparticles synthesized on an Au(111) single crystal surface (see methods section). Figure 2 nanoparticle  induced in the experiment by dosing H2 gas at elevated pressure and temperature as presented in ref. 50 (see “Methods” section). The resulting r-MoS2 particle morphology is represented by a truncated triangular shape, which is bounded by two different types of edges referred to as Mo- and S-edges, respectively. The hydrogen activation leads to a reduced overall 50% S-coverage on the Mo-edge, represented by S monomers located at a bridge position between edge Mo atoms50 . Figure S on these specific sites obtained by a direct counting of missing S atoms in all our STM images. Here, the most probable S vacancies are located at the A positions, with a VS probability at that site being 0.25 . It is slightly less probable to find S vacancies at M positions, with 0.18 of all M sites containing a VS. The number of S vacancies observed directly at the corner site between the Mo-edge and S-edge is considerably lower at 0.06, indicating a higher initial stability of the terminal S monomer at the corner site.Importantly, our analysis shows that the specific Vons Fig.  to identS formation. The model chosen to represent experimentally observed MoS2 nanoparticles was a freely standing truncated MoS2 nanoparticle exposing a short S-edge with 100% S and a five S-monomers long Mo-edge (Mo-5S) comprising of sites of the C, A, and M type   on the C, A, and M locations of the MoS2 nanoparticle is endothermic in all cases. However, the vacancy formation on the corner C site is significantly more unfavorable (ES = 1.97 eV) than the A or M position . For reference, our corresponding calculation of ES on a semi-infinite stripe model gave a value of 0.99 eV, which is slightly lower than that of the M position. To quantitatively account for the effect of temperature and partial pressures of H2 and H2S in the experiment, we calculated the Gibbs free energy of VS formation from which the vacancy fraction at each of the three positions considered can be calculated (see “Theory methods”). We note that some H2S is present in the background gas even if pure H2 is used, due to residual gas and the fact that H2S is released during S vacancy formation, corresponding to a H2/H2S ratio of approximately 103–104 or higher . Figure −4 mbar as a function of the relative partial pressure of H2 and H2S from sulfur rich (H2/H2S > 1) to sulfur poor conditions (H2/H2S > 107). The model predicts a significant population of S vacancies at the M and A positions in the range of H2/H2S ratio between 103 and 104, in agreement with our experimental findings  from this point leads to almost unity vacancy fraction reflecting fully stripped M and A sites equivalent to a naked Mo-edge, which we never observed experimentally. On the other hand, the model predicts a very small fraction of corner (C) vacancies, below 10−7 as opposed to ~6% found in experiments. This difference may be due to the intrinsic errors in DFT (of 0.1–0.2 eV), which can introduce uncertainties in the calculated vacancy population. Furthermore, while the Au substrate is expected to not significantly influence ES on the M and A sites, which is captured well in semi-infinite models50, its influence on the corner site could be a source of error in our calculation of vacancy coverage for C positions in Fig. S formation energies on C sites are dependent on the edge length. Figure ES) at the corner site  at room temperature (300 K). After exposure to thiophene, the r-MoS2 nanoparticles do not reveal any interaction with thiophene on the basal plane, in accordance with the expected chemical inertness of the MoS2(0001) basal plane. On the longer Mo-edge, however, we observe a changed structural pattern indicative of thiophene adsorption, where the S in thiophene is expected to adsorb at the sulfur vacancy. For example, the edge protrusions indicated by yellow arrows at a C and A site on the MoS2 nanoparticles in Fig. S observations before (black bars) and after dosing thiophene molecules at 10−6 mbar at 300 K, 400 K and 650 K substrate temperatures, respectively, is depicted in Fig. S fraction drops from 0.06 to less than 0.03. These changes demonstrate that thiophene molecules can fill the C and A vacancy sites at the Mo-edges already at room temperature without a substantial activation barrier. Dosing thiophene at 400 K shows a further decrease in the vacancy fraction for all the positions (yellow bars). At this temperature, the corner sites seem to be almost fully occupied by thiophene molecules. The vacancy fraction at A still decreases more rapidly than at position M, suggesting that the inhibition of thiophene adsorption at position M may be related to larger steric effects of thiophene adsorption at these sites. Experiments carried out at even higher temperatures  among the three site types close to the initial values. Since high temperature alone is not enough to desorb the thiophene  of Supplementary Table 2 dimer can also be formed on the other side of the edge closer to the C site, in a configuration with an exothermic BE of −0.39 eV (note here we used a six S-atom-long Mo-edge (Mo-6S)).The most stable adsorption mode found for thiophene on the A vacancy site is shown in Fig. mentclass2pt{minimmentclass2pt{minim2 or M and S2, respectively, in the lower part of Fig. 2 dimer is located at the high contrast protrusion on only one side and the dark spot reflects the rather open region between thiophene and the S2 dimer seen in all cases in the structures in Fig. 2 dimer is located either away from the nearest corner site or towards the corner position. We also observe the modeled displacement of the corner S monomer from a bridge to on top in configuration 4b, as indicated by the position of the protrusion at the green arrow in the superimposed image below Fig. The STM images in Fig. 2 dimerization, per se, is typically endothermic with energies ranging from 0.7–1.9 eV depending on the location of the dimers and the concomitant vacancy  is considered to take place by thiophene adsorption, subsequent S hydrogenolysis, followed by VS regeneration and H2S release. A favorable reaction site should thus exhibit an appropriate balance between a favorable adsorption energy of thiophene (BE) and a reasonable VS formation energy (ES). Our findings indicate that corner sites in a MoS2 nanoparticle are very favorable for thiophene adsorption, but our modeling also shows that that vacancy formation is so energetically expensive that a very low fraction of the corner (C) sites will contain a VS under steady-state reaction conditions. This is in line with recent findings on corner site reactivity in Co promoted MoS235. The HDS activity in thiophene DDS is therefore mainly ascribed to Mo-edge sites. We find that VS formation is less expensive on the M and A sites of the Mo-edge, and both sites are shown to be able to adsorb thiophene. Importantly, thiophene adsorption itself leads to restructuring of the S atoms on neighboring sites around the initial VS sites and the formation of a more open site, in some cases reflecting a double VS site. This can either happen by formation of a neighboring S2 dimer, if the starting point is a single VS, or by merger of two next-nearest neighbor VS. The observations illustrate that adsorption of S-containing molecules on the edges does not follow a simple Langmuir (checker-board) adsorption model, but should rather be considered a case where the adsorption site dynamically adjusts to the adsorbate by S displacement. We expect that these dynamic arrangements are relevant in general for HDS reactions of a wider range of compounds, as they explain why there is “enough room” for molecules such as dibenzothiophene to adsorb and undergo DDS on the edges of MoS2. The observations reported here, therefore, have profound implications on our understanding of the reactivity of MoS2-based hydrotreating catalysts, in particular for HDS and hydrodenitrogenation of complex refractory feedstock composed of a blend of large S and N containing compounds as well as for emerging technologies such as hydrodeoxygenation of lignocellulosic biomass.In the HDS catalytic cycle, the overall DDS reaction on a sulfur vacancy site (V2 on Au(111)53. The synthesis of MoS2 nanoparticles on Au(111) was carried out on a clean Au(111) single crystal in a standard ultra-high vacuum (UHV) equipped with a homebuilt Aarhus-type variable temperature STM. The sample temperature was measured with a K-type thermocouple in contact with the backside of the Au crystal. MoS2 nanoparticles were synthesized by physical vapor deposition of Mo evaporation onto the gold surface using an e-beam evaporator (Oxford Applied Research EGCO-4). H2S gas  was dosed for sulfidation corresponding to a background pressure of 1.0 × 10−6 mbar for 15 min, while the sample temperature was 400 K. In order to obtain full crystallinity of the MoS2 nanoparticles, the samples were post-annealed in H2S atmosphere to 673 K for 10 min. The MoS2 nanoparticles were subsequently activated under reducing conditions by back-filling the UHV chamber with H2 50. The reduced (active) MoS2 nanoparticles (r-MoS2) used as the starting point of the experiments reported here were obtained by annealing the fully sulfided samples to 673 K in of 10−4 mbar of H2. For thiophene  dosage, the liquid was kept in a glass container and admitted to the UHV chamber through a leak valve connected to a stainless steel tube directed to the sample surface. Prior to dosing, the thiophene liquid was purified by several freeze-pump-thaw cycles to remove dissolved gas. The gas purity was checked and H2S and H2 partial pressures were measured by a quadrupole mass spectrometer . The samples were exposed to thiophene at three different substrate temperatures: 300, 400, and 650 K; a background pressure of 1.0 × 10−7 mbar was used for 5 min during the thiophene dosage. After each thiophene exposure, the sample was cooled down to room temperature and transferred to the STM for a local inspection. STM images were recorded at room temperature using etched W tips in the constant current mode. The tunneling parameters are noted in the image text as Vt (tunneling bias) and It (tunneling current).The experimental approach is based on the synthesis method of well-defined nanocrystals of MoS2 with Mo-edge containing two to six molybdenum atoms while the S-edge contains four atoms, reflecting the typical truncated triangular shape of a single-layer particle observed in STM experiments. Two layers of sulfur atoms sandwich the molybdenum layer such that they are in the trigonal prismatic positions characteristic of the 2H phase of MoS2. Further, most of the calculations involve a Mo-edge of six molybdenum atoms , also consistent with our observations that metal edges are longer than the sulfur edges. All figures in this document with MoS2 structures and adsorption configurations show Mo atoms in blue, S atoms in yellow, C atoms in brown, and H atoms in gray. The Au(111) support was not included in these calculations; while the support can influence the stability of the edges, the single-layer stripe model used in periodic calculations have successfully captured the correct edge morphologies under sulfidation and reducing conditions, as inferred from STM studies We, therefore, suggest that the given model captures the local electronic structures adequately to provide a comparative analysis between different locations along the periphery of the MoS2 nanoparticles used in experiments. We further assume that the sulfur edge is 100% S-decorated while the metal edge is 50% S-decorated, consistent with ab initio phase diagrams and STM observations50. Importantly, we introduce S-vacancies on the Mo-edge on the center (C), adjacent (A) and middle (M) positions, as needed, to elucidate the energetics of S-vacancy formation and of thiophene adsorption on these vacancies.All calculations (unless specified otherwise) were carried out for a single-layer hexagonal nanoparticle model of MoS55, a plane wave periodic DFT code. Generalized gradient approximation and projected augmented wave (PAW) potentials56 were used with PBE exchange correlation functional57 and the D3 Grimme dispersion correction58. All calculations were carried out in a box that had at least 10 Å of vacuum between two images in all directions. Spin polarization is included in all calculations: the effect of spin was found to be negligible (~0.01 eV on the vacancy formation energy). Plane wave and density wave cutoffs of 400 and 645 eV were used, respectively. A Gaussian smearing of 0.05 eV was used, and the energies were extrapolated to 0 K. Only gamma-point sampling was used in view of the large dimensions of the supercell. The convergence criterion for geometric relaxation was set to 0.02 eV/A.The calculations were carried out with VASPES, is given byENP,with CUS is the energy of the nanoparticle with coordinative unsaturation (CUS or vacancy), 2S in the gas phase, ENP is the energy of the nanoparticle at the equilibrium termination , and 59. The entropy of surface S atoms was assumed to be of vibrational origin while rotational and translational components from statistical mechanics for an ideal polyatomic species were additionally included for gaseous molecules. Vibrational entropy was computed using the Harmonic approximation, which is sufficient for all systems considered here. The binding energy of thiophene, BE, is given byENP, with CUS + Thiophene is the energy of the nanoparticle with thiophene adsorbed on the vacancy (with and without sulfur rearrangements), ENP, with CUS is the energy of the nanoparticle with the vacancy (without rearrangements), and EThiophene is the energy of gas-phase thiophene.The energy of vacancy formation, 59. We start with a 50% S covered Mo-edge and create a single vacancy in the middle of the edge to represent an internal vacancy (corresponding to an M vacancy site in the main text). The supercell was periodic in one direction  where an Mo-Mo distance of 3.19 Å was set based on our optimized lattice constant; the cell had more than 15 Å of vacuum in the other directions. The k-point sampling was 1 × 2  x1 based on the Monkhorst Pack method60. All other settings for these DFT calculations were the same as above. The transition states were calculated using the climbing image nudged elastic band method61 such that the forces on the images were less than 0.1 eV/Å.For transition state calculations in Supplementary Table 1, a periodic single-layer stripe model with four rows of six Mo atoms each and commensurate S atoms was created based on our previous workSupplementary InformationPeer Review File


WIPI proteins (WIPI1-4) are mammalian PROPPIN family phosphoinositide effectors essential for autophagosome biogenesis. In addition to phosphoinositides, WIPI proteins can recognize a linear WIPI-interacting-region (WIR)-motif, but the underlying mechanism is poorly understood. Here, we determine the structure of WIPI3 in complex with the WIR-peptide from ATG2A. Unexpectedly, the WIR-peptide entwines around the WIPI3 seven-bladed β-propeller and binds to three sites in blades 1–3. The N-terminal part of the WIR-peptide forms a short strand that augments the periphery of blade 2, the middle segment anchors into an inter-blade hydrophobic pocket between blades 2–3, and the C-terminal aromatic tail wedges into another tailored pocket between blades 1–2. Mutations in three peptide-binding sites disrupt the interactions between WIPI3/4 and ATG2A and impair the ATG2A-mediated autophagic process. Thus, WIPI proteins recognize the WIR-motif by multi-sites in multi-blades and this multi-site-mediated peptide-recognition mechanism could be applicable to other PROPPIN proteins. WIPI proteins are mammalian PROPPIN family phosphoinositide effectors that recognize a WIPI-interacting-region (WIR)-motif to recruit other regulators during autophagosome formation. Here, the authors combine structural and functional studies and present the crystal structure of human WIPI3 with a bound ATG2A WIR-peptide and observe that the WIR-motif binds to three sites in blades 1-3 of WIPI3 and mutating these binding sites impairs the ATG2A-mediated autophagic process. Macroautophagy  is characterized by a double-membrane structure termed autophagosome that captures a variety of cellular components and delivers them to lysosome for degradation4. Formation of autophagosome at intracellular membrane contact sites strictly depends on phosphatidylinositol 3-phosphate (PI(3)P) that can be decoded by a number of specific downstream effectors6. These phosphoinositide effectors cannot only sense the PI(3)P signal but also act as scaffolds to recruit other proteins or protein complexes for the progression of autophagosome formation9. Mutations of these proteins severely impact on the autophagic process and are also intimately coupled with diseases such as cancers and neuronal disorders12.Autophagy is a lysosome-mediated catabolic process for the clearance of damaged organelles or protein aggregates to maintain cellular homeostasis14. There are three PROPPINs in yeast  and four in mammals , WIPI1 to WIPI4)16. PROPPINs adopt a seven-bladed β-propeller fold, in which the two neighboring sites in blades 5–6 contribute to the binding of phosphoinositides19. PROPPINs can also function as scaffolds to integrate the phosphoinositide signaling with other regulators for controlling autophagy. In mammals, WIPI4 forms a complex with ATG2A/B that may act as a tethering factor for autophagosome expansion21, while WIPI2 interacts with ATG16L1 for the efficient recruitment of the ATG12-ATG5-ATG16L1 complex and the lipidation of LC39. Similar scaffolding functions were reported for yeast PROPPINs Atg18 and Atg21, respectively24. In addition, WIPI3 and WIPI4 can act as scaffolds to further integrate the LKB1-AMPK-TSC signaling for controlling the autophagic process7. However, the mechanism underlying the scaffolding role of PROPPINs and their recognition of target proteins remains largely unclear.PROPPIN (β-propellers that bind polyphosphoinositides) proteins are phosphoinositide effectors that play prominent roles in autophagosome biogenesis21. We denote this linear peptide sequence as the WIR (WIPI-interacting-region)-motif. In this study, we determine the structure of WIPI3 in complex with the WIR-peptide from ATG2A. Unexpectedly, the WIR-peptide wraps around the WIPI3 β-propeller and binds to three sites in blades 1–3. Based on the WIR-peptide complex structure, we define the peptide-binding sites in WIPI proteins and the signature WIR-motif in their target proteins. Mutations in these peptide-binding sites disrupt the interactions between WIPI3/4 and ATG2A and impair the ATG2A-mediated autophagic process. This work reveals the multisite-mediated peptide-recognition mechanism of WIPI β-propellers, which could be extended to other PROPPIN family members.Among four WIPI proteins, WIPI3 and WIPI4 specifically recognize a linear characteristic peptide sequence from ATG2A/B20, WIPI3 exhibited excellent protein sample quality and was chosen for biochemical and structural studies25. On the other hand, the WIPI-interacting region in ATG2A was narrowed down to residues 1358–140421. Based on the primary sequence analysis of ATG2, we removed the N-terminal 15 residues from this region and used the two fragments of ATG2A (the long fragment (1358–1404) and short fragment (1374–1404), referred to as ATG2Along and ATG2Ashort, respectively) tagged with GB1 for the isothermal titration calorimetry (ITC) assay   , with deletions of two flexible loops (Blade 2 B–C loop (75–80) and Blade 6 C–D loop (264–281)), shows enhanced protein sample quality and has been crystallized for structural determinationThe complex structure was determined by the molecular replacement method and refined to 2.2 Å  Fig. . The β-pThe interaction interface between the WIR-peptide and WIPI3 can be divided into three sites  Fig. . In N-siTo evaluate the essential roles of these three sites in WIPI3 for recognizing the WIR-peptide, we made point mutations in each site and performed the ITC assay to measure the binding affinities between the mutants and WIR-peptide. All the point mutations were introduced in WIPI3-Δloop because we hardly obtained sufficient amount of full-length WIPI3 mutants for the binding assay , but upon binding of the WIR-peptide, the A–B loop of blade 2 in C-site underwent local conformational changes  are essential for WIPI4 to interact with ATG2A7 Fig. . We furt7 Fig. , confirm7 Fig. . More im7 Fig. . As afor7 Fig. . Thus, i27. Among these mutations, three point mutations  are located in peptide-binding sites  have been found to be linked with human diseasestes Fig. . To evaltes Fig. , support20. Consistent with this, the critical residues responsible for the formation of three peptide-binding sites in WIPI3 are highly conserved in WIPI4  for recognizing the aromatic tail of the WIR-peptide are not conserved in WIPI1 and WIP2  to somewhat mimic the potential target-binding sites in WIPI1-2 . Interestingly, the mutations of these residues had different impacts on the binding to WIPI3. Specifically, the V1380A (rather than V1380Q) mutation affected the binding, while the T1382Q (rather than T1382A) mutation impacted the binding  that form a sharp turn structure Fig. , which in-Φ-X-Φ-X-X-X-Ψ-F” (Φ is a hydrophobic residue and Ψ is an aromatic residue), which is highly conserved in ATG2 family members , suggesting that WIPI proteins may also play an essential role in the linkage of membrane trafficking to autophagosome formation. Nevertheless, the potential binding partners in Supplementary Table Based on all these mutational studies, the WIR-motif for binding to WIPI3 and WIPI4 can be defined as the signature sequence of “β-strand-(X)ers Fig. . In the 20. As expected, mutations in each part of the WIR-motif in ATG2A  decreased the binding to WIPI4  cells with depletions of both ATG2A and ATG2B and stable expression of GFP-tagged LC3 and also checked the autophagic flux with the presence of bafilomycin A1 (an inhibitor of autophagosome–lysosome fusion). Consistent with recent studies29, depletions of ATG2 proteins resulted in the abnormal accumulation of large LC3-positive structures under nutrient-rich condition 7. Structural studies of the WIPI4/ATG2A complex demonstrated that this complex adopts a rod-shaped structure with two opposite tips tethering two different membranes P-containing and PI(3)P-free membranes)21. Meanwhile, ATG2 proteins were also found to contain the lipid-transfer capacity to mediate the lipid-transfer between two membranes30. In this study, we found that the WIR-motif in ATG2A is responsible for binding to WIPI4 and mutations of the WIR-motif disrupted the interaction between WPI4 and ATG2A and impaired the ATG2A-mediated autophagic process   host cells at 16 °C. The GB1-His6-tagged fusion proteins were purified by Ni2+-Sepharose 6 Fast Flow  affinity chromatography using the wash buffer  and elution buffer . The eluted proteins were further purified by size-exclusion chromatography . For WIPI3 proteins, after cleavage of the tag, the resulting proteins were purified by another step of size-exclusion chromatography with the buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT. For ATG2A fragments, recombinant proteins were purified in the same buffer without cleavage of the tag.DNA sequences encoding human WIPI3, WIPI3-Δloop, and two ATG2A fragments (1358–1404 and 1374–1404) were each cloned into a modified pET32a vector. The generation of the fusion construct of WIPI3-Δloop with ATG2A(1374–1404) and the mutations in WIPI3 and the ATG2A fragment were performed by using the standard PCR-based mutagenesis method and confirmed by DNA sequencing. All the primers used in the study were listed in Supplementary Table ITC assay was performed using a MicroCalorimeter ITC200  at 25 °C. Prior to the experiment, the protein samples were dialyzed in the buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. WIPI3 proteins were put in the sample cell and GB1-tagged ATG2A fragments were in the syringe of the instrument. In each experiment, the ATG2A fragment was sequentially injected into the stirred calorimeter cell initially containing the WIPI3 protein sample with the injection sequence of 20 × 2 µl at 2-min intervals. The heat of dilution obtained by the titration of the ATG2A fragment into the buffer was subtracted. The integrated, corrected, and concentration-normalized peak areas of the raw data were finally fitted with a model of one binding site using ORIGIN 7.0 (OriginLab).31, and were processed and scaled using HKL200032. The structure were determined by the molecular replacement method with the structure of WIPI3 (PDB code: 6IYY [10.2210/pdb6iyy/pdb]) as searching models using PHASER33. The WIR-peptide was manually modeled into the structure according to the 2Fo-Fc and Fo-Fc electron density maps using COOT34. The structures were further fitted and refined with PHENIX35. The structure figures were prepared with the program PyMOL. The statistics for data collection and structural refinement were summarized in Supplementary Table Purified WIPI3 proteins were concentrated to ~20 mg/ml in the buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, and 50 mM DTT for crystallization. Crystals of the WIPI3-Δloop/WIR-peptide complex (the WIR-peptide-WIPI3-Δloop fusion protein) were grown in 0.1 M Tris-HCl, pH 8.5, and 20% (v/v) Ethanol and were obtained using the vapor diffusion method (sitting drop) at 16 °C. Before being flash-frozen in liquid nitrogen, crystals were cryo-protected in their mother liquor supplemented with 16% (v/v) ethylene glycol. Diffraction data were collected at the beamline BL19U at the Shanghai Synchrotron Radiation Facility (SSRF) using with a wavelength of 0.979 Å at 100Kg for 30 min at 4 °C to separate soluble fractions and cell debris. Supernatants were applied to Strep-Tactin Sepharose beads (IBA) and incubated for 1 h at 4 °C. The beads and non-bound proteins were separated by centrifugation at 1000 g at 4 °C. After washing the beads three times with a wash buffer composed of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM DTT, 1 mM EDTA, and 0.2% (v/v) NP-40, the bound proteins were eluted with the elution buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM DTT, and 5 mM desthiobiotin (IBA). The eluted products were analyzed by SDS-PAGE. GFP-tagged WIPI4 and TSF-tagged ATG2A were detected by western blot using the anti-GFP  and anti-Flag  primary antibodies, respectively, and the goat anti-mouse secondary antibody . Overall 1% of the input was loaded onto SDS-PAGE as the loading control and detected by the anti-beta-actin antibody .GFP-tagged WIPI4 and Tandem-Strep-Flag (TSF)-tagged ATG2A (wild-type or mutants) plasmids were co-transfected into HEK293T cells  using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cells were collected 48 h after transfection. Cell pellets were lysed in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM DTT, 1 mM EDTA, 1 × Protease inhibitor cocktail, 1 mM PMSF, and 1% (v/v) NP-40. Lysates were centrifuged at 20,000 ATG2A/ATG2B and stably transfected with GFP-LC3 were generous gifts from L.Y., Tsinghua University. DNA sequences encoding full-length ATG2A and its various mutants were cloned into the pCAG-Tandem-Strep-Flag vector. Cells were cultured in DMEM (GIBCO) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin and were grown at 37 °C in a humidified atmosphere containing 5% (v/v) CO2. Cells were transfected with wild-type ATG2A and various mutants by the electroporation apparatus (Bio-Rad) according to the manufacturer’s instructions. After 48 h, cells were washed three times by PBS and fixed by 4% (w/v) paraformaldehyde. For bafilomycin A1 treatment, cells were cultured in the complete medium containing 10 nM bafilomycin A1 for 2 h before being fixed. ATG2A and its various mutants were detected by immunofluorescence with the anti-Flag primary antibody  and dylight 594 secondary antibody . Fluorescence images were obtained on an inverted confocal microscope  equipped with a 100 × oil-immersion objective lens (NA = 1.30). Confocal settings used for image capture were held constant in comparison experiments. All the fluorescence images were processed and analyzed using the ZEN imaging software (Carl Zeiss). Microsoft Excel and GraphPad Prism were used for statistic analysis.NRK cells with double knock out of Further information on experimental design is available in the Supplementary InformationReporting summary


Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.The springtail,  Megaphorura arctica Tullberg 1876, is widely distributed in the northern parts of the Palaearctic region where it is common and abundant along sea shores1. Aggregations of the species are often found in decaying seaweed of the supra-littoral zone, under stones and in moss below bird colonies3. Measurements of the winter temperature of its habitat show that the species in the field is able to survive temperatures of −20 °C or lower4. Freeze-tolerance  has never been observed in springtails, and it is therefore generally accepted that they belong to the freeze-avoiding species6. As the name of this overwintering strategy indicates, these species have physiological adaptations enabling them to avoid internal ice formation even though the ambient temperature for long periods can be much lower than the melting point of their hemolymph. However, springtail species have followed two fundamentally different trajectories of adaptation to subzero survival by freeze-avoidance. Species living predominantly on the ground surface or in vegetation (epigeic species) have relatively impermeable cuticula and are typical freeze-avoiders with high capacity for supercooling similar to many insects7. Other springtails inhabiting deeper layers of the soil (hemi- and eu-edaphic species), have little cuticular resistance to desiccation, and base their freeze-avoiding capacity on an alternative strategy termed cryoprotective dehydration. In this strategy, the difference in water vapor pressure between ice in the soil and the supercooled hemolymph drives a net outflux of water vapor9. The force of this vapor pressure difference is so large that even a few degrees of supercooling will result in substantial water loss, continuing until the vapor pressure of body fluids equals that of the surrounding ice10. At this stage, the risk of ice formation in the body has been eliminated, and subzero survival is ensured.The springtail, M. arctica on a seasonal timescale efficiently adjusts the melting point of its hemolymph to equal the temperature of its winter habitat8. However, in the initial phases of cryoprotective dehydration, melting point depression rates can be slow with the result that hemolymph at this stage is supercooled by a few degrees until water contents have decreased to levels where further evaporative water loss produces higher rates of melting point depression11. Even though supercooling is limited to a few °C, and of short duration, physical contact between springtails and ice crystals in the habitat potentially may result in inoculative spread of external ice to hemolymph through hydrophilic surfaces or openings of the animal such as mouth or ventral tube. These considerations prompted us to look for antifreeze proteins (AFPs) which are well-known for their ability to limit ice growth in freeze-avoiding fish and insects14.Studies have shown that Hypogastrura harveyi, present in Eastern Ontario15. Extracts of active H. harveyi, collected from snowbanks in late winter, were able to depress the freezing temperature by 5.8 °C below the melting point. The proteins responsible for this strong thermal hysteresis (TH) activity were unlike any previously described AFPs from fish, insects, plants and microorganisms, being rich in glycine and having tandem tripeptide repeats of Gly-aa2-aa3, where aa2 was frequently also Gly. As predicted by Lin et al.16, these glycine-rich tracts folded as polyproline type II helices linked by turn regions17. The small (6.5 kDa) isoform of H. harveyi AFP (HhAFP) had six of these helices forming two sheets secured by intrachain hydrogen bonds in the glycine-rich core, and by two disulfide linkages. A larger isoform (15.7 kDa) was predicted to fold in a similar manner but with 13 polyproline type II helices, again packed into two sheets with a glycine-rich core18.An AFP was previously characterized from a springtail species, M. arctica for AFPs is the expectation of finding additional novel ice-binding proteins in different Collembola families that might elucidate structure-function relationships in AFPs and the mechanism by which these proteins bind to ice19. Four different AFP types have been found in teleost fishes14. Several different types have been characterized in insects; and those described to date in plants and microorganisms have added to the remarkable diversity of ice-binding proteins. It is thought that the variety in fish AFPs reflects the relatively recent impact of sea-level glaciation in the Cenozoic era that challenged teleosts to develop ice control at a late stage of their radiation when extant families already existed21. Collembola are considerably older than teleost fishes, widespread on all continents, including regions where there are sub-zero temperatures, and might therefore yield a similar diversity of AFPs. Indeed, preliminary characterization of Gomphiocephalus hodgsoni from Antarctica suggests it contains an AFP with a distinct amino acid composition that is rich is Cys and His22.Another reason to probe M. arctica (2.3 mg) with buffer (18.4 µL) categorically showed the presence of an AFP in the crude homogenate based on ice crystal shaping and high thermal hysteresis activity  with buffer (50 mL) was performed to obtain enough AFP for biochemical characterization. The AFP was isolated through four cycles of rotary ice-affinity purification with the emphasis placed on purity rather than yield. Thus, the ice shells were rinsed before being melted and re-extracted; and no back-extractions were performed on the excluded liquid fractions to recover any AFP that was left behind. When the concentrated final ice fraction (500 µL) was tested, it had a thermal hysteresis activity of 3.9 °C, with ice crystal shaping and growth characteristics , where the 6.5-kDa isoform had 48 mol% Gly and 14 mol% Ala. The dominance of Gly and Ala also suggested that the several major peaks seen in the MALDI mass spectrum are isoforms of MaAFP rather than impurities. Both purified MaAFP and the HhAFP isoforms had few of the larger amino acids, such as the aliphatic residues  and aromatic residues . The somewhat higher combined level of these residues (18%) and lower level of Gly in the ice affinity-purified MaAFP material, compared to the defined sequences, could be due to small amounts of contaminating proteins.Analysis of the concentrated pure extract by MALDI mass spectrometry showed the presence of several low molecular weight proteins Fig. . The firMaAFP amino acid composition  encouraged us to digest the protein with trypsin and sequence the resulting peptides  in the NCBI database25 showed significant similarity to any of the MS/MS sequences , helix 4 was rotated relative to the final model with the Ala sidechains directed inwards and the Gly residues surface exposed. This was easily corrected using the program Modeller so that all residues larger than Gly, within the six helices, are now surface exposed  is partially exposed and a small (0.4 Å) shift of the backbone was adequate to prevent steric clashes  or Pro (dark grey). The turns at both ends are also somewhat variable, but the two disulfide bonds  and ribosomal protein L35 (RPL35)) were compared, the diagonal lines spanning the coding regions indicate that these sequence segments are indeed well conserved Fig. . Howeverern Fig. . One intIF4 Fig.  correspo30. For instance, AFP-mediated freezing point depression in the spruce budworm (Choristoneura fumiferana) of 6 °C is an important component in overwintering of first-instar larvae remaining unfrozen and viable in the boreal forest at temperatures of −20 to −30 °C28. Despite M. arctica’s reliance on cryoprotective dehydration, and not deep supercooling, as its main strategy for avoiding freezing, these springtails also possess a potent AFP that complements the set of adaptive traits for winter survival in this species.Examples of highly potent AFPs are mostly found in freeze-avoiding insects with high supercooling capacityMaAFP can afford its host. However, since we know the dry weight of the animal sample and how much buffer was used for the crude extract measurements, we can make an estimate of this. We made an extract from 2.3 mg dry M. arctica tissue in 18.4 µL of buffer, equivalent to a water content of ca. 7.8 mg water mg−1 dry tissue, which is at least twice the normal water content of fully hydrated M. arctica32. It is therefore likely that the freezing point depression provided by MaAFP in vivo is similar or perhaps even higher than the 3.1 °C measured for the crude extract.Due to logistic limitations we were not able to measure antifreeze hysteresis activity of hemolymph samples from live specimens in the field, and we therefore do not have a direct determination of the amount of freezing point depression 33. At this stage, there is a risk that external ice will spread to the animal causing inoculative freezing. Until now, we have been missing a good explanation how inoculative freezing is avoided in frozen soil, but the discovery of a potent AFP in M. arctica closes this gap in knowledge. Thus, the freezing point depression that we estimated to ca. 3 °C seems to meet with the needs outlined above. Studies of the exact location of MaAFP in the animal would further explain the ability to avoid inoculative freezing. AFP can be expressed in various tissues of insects such as the midgut, hemolymph and epidermis35. Expression of AFP in epidermis cells could be particularly relevant for inoculative freezing in M. arctica and other springtails using cryoprotective dehydration34.Since cryoprotective dehydration can sometimes be a slow process some supercooling capacity  is needed in the initial phases of dehydration, especially if the cooling rate is highTetracanthella wahlgreni to −3 °C in close contact with ice caused inoculative freezing and death even though supercooling points of this species ranged between −15 and −25 °C if specimens were not in contact with external ice36. Since contact with ice is decisive, the risk of inoculative freezing is highest when soil moisture content is high38. Interestingly, several hygrophilic soil invertebrates  survive sub-zero temperatures by freeze-tolerance, or can switch between cryoprotective dehydration and freeze-tolerance depending on the moisture conditions of their overwintering habitat41. This dual strategy is consistent with the fact that both freeze-tolerance and cryoprotective dehydration involves tolerance of cellular dehydration and the physiological adaptations connected to this11.Inoculative freezing may be a significant cause of lethality for springtails and many other soil living invertebrates that do not tolerate freezing of their body fluids. For example, Sømme and Conradi-Larsen (1977) observed that cooling the springtail MaAFP has the attributes of a hyperactive antifreeze protein in having high thermal hysteresis activity at low protein concentrations, with evidence of AFP binding to the basal plane. This is typical of arthropod AFPs, which have generally been selected for higher freezing point depression activity than the moderately active AFPs in fish, which never face temperatures lower than −1.9 °C24. MaAFP is made up of what appear to be several isoforms. Multiple AFP isoforms are common in fish45 and arthropods47. They arise from gene duplication followed by divergence. Since freezing point depression is a function of AFP concentration, AFP-producing organisms seem to be under selective pressure to increase the amount of circulating AFP by gene amplification48. For AFPs made from repetitive sequences it is easy to generate length variation by expansion and/or contraction of the repeats. Examples of this can be seen in AFPs from the beetles Tenebrio molitor and Dendroides canadensis where the repeating unit is a 12-residue coil in a beta-solenoid structure. Common isoforms have seven coils but some have eight49. Another example of length variation is in the moth, Choristoneura fumiferana, where one isoform has two extra 15-residue coils in its beta-solenoid structure50. Interestingly, these larger isoforms are more active than the shorter ones because the ice-binding site is increased in area. Thus, there may also be selective pressure to increase isoform size.HhAFP and MaAFP seems feasible too, since they are based on Gly-aa2-aa3 tandem tripeptide repeats. However, unlike the single solenoid structures of the beetle and moth AFPs, the polyproline type II coils must pack into a higher order tertiary structure where the coils are bundled together to form a Gly-rich core. Thus, the expansion of a single coil would potentially destabilize the overall structure. Instead, an increase in isoform size in HhAFP seems to have been accommodated by an increase in the number of coils from 6 to 13, and with it again a considerable increase in antifreeze activity18.Lengthening of the polyproline type II coils in MaAFP and HhAFP it is not possible to unequivocally say they are descended from a common ancestor sequence. There are well-documented examples of simple repetitive AFPs that have independently originated from different progenitors. For example, the antifreeze glycoproteins (AFGPs) of Antarctic Notothenioids and Arctic cods are made up of a tandemly repeated Ala-Ala-Thr tripeptide with a disaccharide O-linked to the Thr45. Although these AFGPs are virtually indistinguishable and share almost complete sequence identity as a set of isoforms of different lengths derived from polyprotein precursors, they are not homologs. The Notothenioid AFGPs have arisen from an intronic sequence from the gene for the pancreatic enzyme, trypsinogen52. AFGPs of the northern cods, however, are derived from a different DNA sequence53. Type I AFPs are another example of convergent evolution to a structural and functional commonality. These Ala-rich, alpha-helical AFPs, with an underlying 11-amino-acid repeat, have independently arisen at least four times in different branches of teleost fishes from unknown precursor sequences54. Since they are 60–65% Ala they all have >25% sequence identity, which is the measure applied to recognize homology. Clearly this criterion is invalid when dealing with proteins that have such a biased amino acid composition.Despite the high sequence identity between MaAFP and HhAFP is more complex because, unlike the AFGP and type AFP I examples above, these AFPs have a higher order of protein structure where six helices of similar length pack together to form a Gly-rich core. In addition, there are four Cys, whose positions are conserved such that the two disulfide bonds they form to help secure the helix bundle structure are in the same places in both proteins. These structural features argue for homology. A comparison of the 5′- and 3′-untranslated regions shows no sign of sequence identity, which could argue against homology. One possible explanation for these confounding results is that MaAFP and HhAFP are ancient homologs that have survived from an earlier ice age, perhaps even before these two species diverged. Collembola originated at least 400 Ma and would have encountered the ice ages of the Permian period ~300 Ma58. If so, some of the extant springtails may have derived their AFPs from this period. Those that retained their AFP genes during the 200+ million years between the Permian and Cenozoic ice ages would have been well equipped to survive in present sub-zero conditions. This hypothesis might explain why the UTRs of the HhAFP large and small isoforms also appear unrelated, as do the UTRs of MaAFP and HhAFP. If the isoforms predate speciation and date back to the Permian period, regions of the gene not under selective pressure would have had ~300 million years to drift. One way to test this hypothesis would be to sample additional freeze-resistant springtail species for the type of AFPs they might possess to see if they too have high-Gly, polyproline type II helical bundles suggestive of an early origin.The comparison between M. arctica were collected in August 2017, on a beach near Sauðárkrókur, north-west Iceland, and brought back to the laboratory in Silkeborg, Denmark. The animals were kept at 5 °C in 200-mL beakers on a substrate of decomposing brown algae. In order to acclimate springtails to winter conditions, they were kept in darkness at 5 °C for three months followed by about two months at 1.5 °C. At this point, ~1,500 adult animals were taken and freeze dried for two days to complete dryness. The dried animals were stored in Eppendorf vials at −80 °C until they were shipped by courier to Queen’s University, Canada.Adult 59 as the depression of the freezing point (°C) below the melting temperature. To establish the presence or absence of TH, a soluble extract was prepared from freeze-dried M. arctica (2.3 mg). The dried tissue was mixed with 18.4 µL of homogenization buffer (50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 2 mM phenylthiocarbamide, 1x cOmplete, EDTA-free protease inhibitor tablet ) and homogenized by hand, using a pellet pestle  in a 1.5-mL tube. The homogenate was centrifuged at 16,000 × g for 15 min and the supernatant tested for antifreeze activity.Antifreeze activity, referred to here as thermal hysteresis (TH), was measured in a nanolitre osmometerM. arctica (0.30 g) were homogenized at 4 °C in 50 mL of the above buffer containing 1 mM EDTA. The homogenate was centrifuged at 31,000 × g for 30 min, and the supernatant was filtered through glass wool to remove lipid. AFP was extracted from the filtered supernatant (<50 mL) using four cycles of rotary ice affinity purification61, but scaled down in size from the previously reported protocol. Thus, the starting ice shells contained 10–15 mL of ddH2O in a 250-mL round bottom flask. The rotating ice-shells were cooled in an ethylene glycol bath for 1.5–2 h starting at −1.5 °C and decreased by 0.2 °C every 0.5 h until ~25 mL of liquid remained in the flask. Once the liquid was ~50% incorporated into the ice-shell the 250 mL flask was removed from the apparatus, the liquid was poured out and diluted to 50 mL with ddH2O, and the ice-shell was quickly rinsed with 5 mL of homogenizing buffer. The ice shell was melted with 5 mL of 10× homogenate buffer containing 10 mM PTU and 10 mM PMSF then diluted to 50 mL with ddH2O. This process was repeated for four rounds of ice-affinity purification. The final melted ice sample was concentrated using an Amicon Ultracel 3 K Centrifugation filter spun in a refrigerated Sorvall ST16R centrifuge at 4000 rpm until a volume of ~500 𝜇L was reached.For a larger-scale extraction, freeze-dried 61.Amino acid composition analysis was performed at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre (SPARC) BioCentre as previously describedMALDI mass spectrometry was performed using α-cyano-4-hydroxycinnamic acid matrix on dried droplets with a SCIEX Voyager DE Pro in Linear mode by the Queen’s Protein Function Discovery Facility. Tandem mass spectrometry protein sequencing of tryptic fragments separated by high-performance liquid chromatography (LC-MS/MS) was done using ESI (nanospray) with an Orbitrap (FT-ICR) by SPARC BioCentre Molecular Analysis .H. harveyi AFP sequences (ABB03725.1 and ADI39904.1) were used as query sequences in BLAST searches  of M. arctica sequences62. Additionally, all 16379 M. arctica ESTs were downloaded from NCBI, and translations of all six reading frames63 were scanned for sequence strings identified by LC-MS/MS. To find control sequences for dot blot comparisons, previously sequenced H. harveyi ESTs for nuclear genes were searched by BLAST64 for two examples that encoded homologous sequences in the M. arctica EST database. These were then assembled into contigs using the Cap3 Sequence Assembly Program65. Dotmatcher http://www.bioinformatics.nl/cgi-bin/emboss/dotmatcher, written by Ian Longden, formerly at the Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK, was used to compare dot plots of the two nuclear gene cDNAs with complete coding sequences, 5′ UTRs ≥ 60 bp and complete 3′ UTRs, as well as cDNAs encoding the AFPs.MaAFP sequence was submitted to Phyre226 using both normal and intensive mode. An alignment sequence of MaAFP against HhAFP was inputted into MODELLER which performed homology modeling to produce the refined model of MaAFP66. GROMACS was used with both the refined model of MaAFP and the crystallographic structure of HhAFP67. The structures were energy minimized after being solvated in a box of water, after which 0.1 ns position-restrained molecular dynamics runs at constant-volume and constant-pressure were done. These were followed by unrestrained molecular dynamics at a temperature of 277 K for 20 ns where temperature and pressure were maintained with the v-rescale and Parrinello–Rahman protocols, respectively.The Supplementary information.


The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylation, highlighting the underappreciated protein-histidine kinase activity of NME family members, specifically in human cells. In parallel, information about the structural and functional aspects of the NME family, and the knowns and unknowns of histidine kinase involvement in cell signaling are summarized. Ubiquitous nucleoside diphosphate kinases (NDPKs) were identified early in the 1950s based on their ability to catalyze the transfer of a phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) in yeast . A few yThis review will discuss the complexity and polyvalence of NDPK activities and the challenges faced in working on this unstable post-translational modification, and outline the progress that has been made in the past few years in understanding how protein-histidine phosphorylation is catalyzed by NME family members and used to regulate cellular functions . NME (for non-metastatic) genes, also called NM23 (for non-metastatic protein 23) genes, and are highly conserved through evolution with multiple synonyms gene names in different organisms, including AWD, NDK, YNK, Gip17/Guk, NmeGp1sd [NDPK enzymes are expressed from NmeGp1sd . This muNmeGp1sd . A wholeNmeGp1sd  that areNmeGp1sd ). There NmeGp1sd ,12 althoNmeGp1sd ,13. NME1NmeGp1sd .The sequence alignment reveals common features, particularly in the catalytic domain, but distinct features in loops and the N- and C-termini. As mentioned above, NME kinases were first associated with a nucleoside diphosphate kinase (NDPK) activity that catalyzed the transfer of the γ-phosphate from nucleoside triphosphates through a phosphohistidine enzyme intermediate to nucleoside diphosphates resulting in the synthesis of nucleoside triphosphates other than ATP ,16,17 F. But sevNME kinases are ubiquitous, but only a few NME partners and protein substrates are currently known. An additional complexity is that NME family members can interact with each other, raising the functional importance of NME oligomerization, that will be also addressed. NME1 was originally defined as the first metastasis suppressor gene, because its RNA level was higher in tumors with low metastatic potential [Disregarding the molecular mechanism, NME members are reportedly involved in multiple physiological and pathological conditions . Indeed,otential . Besidesotential . NME1 isotential , and knootential ,28.Taken together, the publications related to NME and the synonymous NM23 or NDPK, have grown significantly since 1990, with around 1823 publications according to PubMed. Recent progress in elucidating the functions of this intriguing protein family was discussed by experts in the field at the 11th International Congress on the NDPK/NME/AWD protein family in October 2019 in Annecy, France. Their presentations clearly illustrated the complexity mentioned here, with emerging importance for understudied members such as NME3/4/6/7. In this review, we focus on the histidine kinase activity of the NME family, and its role in physiological and pathological conditions related to NME family members.Phosphohistidine (pHis) belongs to the N-phosphate class of SONAtes  and is characterized by an acid-labile and thermosensitive phosphoramidate bond, in contrast to the O-phosphate category, which includes the conventional phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) ,9,29. pHHistidine phosphorylation is particularly well-known in the two-component system (TCS) or multi-component phosphorylation system used in bacteria for cell signal transduction. In TCS signaling, a transmembrane histidine kinase, generally a sensor kinase, is autophosphorylated on a catalytic histidine, and this phosphate is then transferred onto an aspartate in a response regulator protein, which transmits the signal. Histidine phosphorylation is found not only in bacteria, but also in various other organisms such as fungi and plants and more recently has been associated with important mammalian cellular processes ,31.Despite its long history, the mammalian pHis proteome has been difficult to study because of the acid-lability and thermosensitivity of this modification, which is not compatible with most of the standard phosphoproteomic methods used for pSer, pThr and pTyr analysis. Indeed, this is one of the reasons why our knowledge about phosphorylating events that generate phosphoramidate bonds has progressed slowly. The higher free energy of these P-N bonds leads to rapid hydrolysis at low pH or upon exposure to high heat. Even exposure to mild heat and acidic conditions, such as pH = 6.0 and 60 °C for 30 min, as well as certain primary amines, effectively hydrolyzes pHis. It has also been reported that the 1-pHis isomer is less stable than the 3-pHis isomer ,33. Yet, over the last few years, several new tools have emerged and have led to a resurgence in the study of histidine phosphorylation, specifically thanks to the development of histidine phosphate analogs and pHis monoclonal antibodies ,35,36,37These recent studies have begun to highlight the potential of histidine phosphorylation in multicellular organisms as a reversible regulatory protein modification and support a possible role for histidine kinases in cell signaling. NM23 gene products, primarily NME1-4, is seemingly not necessary for all of their biological functions [The intrinsic nucleoside diphosphate kinase (NDPK) activity associated with the unctions . This stE. coli showed that NDP kinase can phosphorylate histidine protein kinases, such as EnvZ and CheA, which both catalyze pHis-pAsp phosphorelay signal transduction systems in bacterial TCS [Arabidopsis thaliana, one report suggested that NME1 phosphorylated the kinase suppressor of Ras (KSR) at Ser392, as well as Ser434 when Ser392 was mutated [Additionally, studies in rial TCS . Similarrial TCS ,53. Addi mutated .Several studies have shown that NME phosphorylates the histidine in the catalytic site of ATP-citrate lyase (ACLY) ,56,57 anInterestingly, NME2 forms complexes with βγ subunits of heterotrimeric G-proteins and contributes to G protein activation by increasing the high-energy phosphate transfer onto GDP via formation of a phospho-H266 intermediate in Gβ subunits . FurtherThese diverse observations are among several types of evidence that support the concept that NME acts as a histidine kinase. However, as will be discussed further, one issue with NME acting as a protein kinase is that structure of its active site is designed to bind nucleotide tri/diphosphates, which might be expected to restrict access of protein histidine residues. Nevertheless, the fact that histidine residues such as H266 of Gβ are exposed on the protein surface, and also the recent finding that the NME1 activator molecule (NMac1) induces NDPK activation through allosteric conformational changes, suggest possible ways in which NMEs could use proteins as substrates ,62.Newly developed tools, such as sequence-dependent phospho-specific NME antibodies and NME inhibitors ,63, willDuring the past 25 years, more than 10 structures of human NMEs have been solved, including crystal structures of NME1, NME2, NME3, NME4, and NME10 . The firHuman NME1-4 are highly conserved (sequence identity > 50%), but relatively distinct from human NME5–10. Since 2002, structures of other human NME subtypes have been solved, showing highly similar architectures compared to NME2 . NME1, 2Human NMEs have been found to be multifunctional enzymes, with activities including NDP kinase, protein kinase, and 3′-5′ exonuclease. Substrates of the NMEs acting as NDP kinases and 3′-5′ exonuclease (NDP and dinucleotide respectively) have been observed bound to the same site of the enzyme (PDB ID: 3BBB ). DespitH. sapiens, M. xanthus, and D. discoideum, etc. Both ADP and GDP substrates bind to the NDP kinases in a similar manner. The active site is located in a cleft formed by two helices (helix 44–52 and helix 60–70). The base of the nucleotide is near the protein surface, exposed to the solvent. More specifically, by using NME2 as an example, F60 of helix 60–70 and V112 of the Kpn loop (94 to 114) form a clamp, stabilizing the base of the substrate  and even extracellular localizations ,105,106.nhibitor . FurtherDrosophila, the equivalent histidine is H119 in the awd gene, which encodes the NME homolog. Loss of function of the awd gene can be rescued by expression of WT AWD but not with the mutated H119A form, showing the importance of the catalytic site in this gene function [NME1/2 null background, re-expression of NME2 H118N catalytic inactive mutant led to lower basal G protein activation compared to the WT form [To understand the biological effects related to NME’s function as a protein-histidine kinase, site-directed mutagenesis of the catalytic site has been performed. Mutations of H118 to prevent its autophosphorylation, which is essential for NME kinase function, have been explored in several models. In function . On an N WT form . This reDrosophila as the “killer of prune” mutation since it was lethal in combination with the prune gene mutation [Because H118 is important for both nucleotide and protein-histidine kinase activity, experiments carried out by mutating only this amino acid are unable to define which activity is responsible for the observed phenotypes. To distinguish between these two functions, the consequences of mutating Pro96 of NME1 has been investigated. Mutation of Pro96 to serine causes conformational changes in NME affecting its binding to protein substrates . This mumutation ,116,117.mutation . Recent mutation . InteresAnother recent paper from the Steeg group reported that P96S mutant NME1 was unable to promote endocytosis or inhibit EGFR activation , demonstWork using NME mutants has revealed how its histidine kinase function is important in cell migration and invasion, in cellular growth and development, endocytosis, and cell signal transduction. As a summary, some NME protein kinase functions are depicted in Histidine phosphorylation can be reversed by phosphohistidine phosphatases, but as is the case for histidine kinases, only a few are currently known. NME2 pH118 is dephosphorylated by Phosphoglycerate Mutase Family Member 5 (PGAM5) . PGAM5 hAnother recently discovered histidine phosphatase is phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), which was first purified from swine brain tissue . AlthougOther phosphatases such as STS-1 and STS-2 are members of the PGAM family and share sequences with histidine phosphatases. Therefore, they are potential candidates to dephosphorylate histidine residues possibly targeted by the NME histidine kinase . Other pWhile many of the molecular functions of NME in the cell remain unknown, recent progress has been made by investigating NME binding partners. To a large extent, NME function and its partners have been studied in the context of cancer, an outgrowth of the fact that NME was first described as a metastasis suppressor gene. Nevertheless, studies in different organisms have revealed that NME is essential for biological activities related to development and other disorders in addition to cancer. Not a lot is known about NME protein kinase activity and cell signaling. Here, we discuss the few instances where it is known that NME regulates signal transduction through histidine phosphorylation. However, most of the following studies relate to NME function and signaling modulation in different models, independent of its protein histidine kinase activity, but it is interesting to reconsider some of them in the light of histidine phosphorylation. NME1 was first described as a metastasis suppressor in a study comparing RNA levels in mouse metastatic melanoma cell lines with non-metastatic cells, which showed that Nm23/NME1 expression was downregulated in the metastatic cells . NME1 isCaenorhabditis elegans Masoudi, et al. [NME has a cell proliferation inhibitory function. To achieve this, NME1 through its protein phosphotransfer function activates KSR by phosphorylation of S392 leading to altered subcellular localization . KSR is , et al. . Deletio, et al. ,152. pMA, et al. . Surpris, et al. ,153. A p, et al. . NeverthAnother signaling pathway where histidine phosphorylated NME might act is through the AKT signaling pathway. In a mouse hepatocellular carcinoma model, levels of activated pS473 AKT were increased in tumors expressing higher levels of NME and its histidine phosphorylated form compared to the normal control . AlthougThere are other important signaling pathways regulated by NME for metastasis inhibition. However, there is no direct link between histidine phosphorylation and NME function in these cases, and it remains to be determined whether histidine phosphorylation might be important in some of them. For instance, silencing of NME increases Rac1 signaling and MAPKs, including ERK, JNK, and PI3K/AKT pathway activation . NME1 alC. elegans, binding of microRNA mir-35 to NDK-1 modulates the activation of the MAPK pathway resulting in DNA damage-induced apoptosis of germ cells [C. elegans and in humans NME promotes the engulfment of apoptotic cells through its interaction with Dynamin1 and 2 [NME1 can also promote cell death or enhance the engulfing phagocytic capacity of cells . In C. erm cells . In a carm cells ,164. In n1 and 2 ,165,166.Is it possible that NME regulates MAPK/AKT signaling mediated cell proliferation, motility, and apoptosis by its histidine kinase activity? Because most of the previous studies did not focus on detecting pHis protein signals, this question cannot yet be answered, and future experiments, including with other NME family members, will be needed. Interestingly, ongoing work presented at the 11th International Conference on the NME gene family (NME 2019 conference) has highlighted a critical function for NME3 in maintaining genome stability and a possible role in breast tumor metastasis . Likewis2+ channel activity [NME1 and 2 and the catalytic H118 residue are highly conserved from yeast to humans ,72. NMEsactivity , which iactivity . Phosphoactivity . Moreoveactivity ,172,173.activity . The samDrosophila has demonstrated that the deletion of the NME homolog awd gene, causes defects in the formation of imaginal discs during the development of larvae to adults [awd plays a role in the migration of cells during tracheogenesis and border cell movement, with awd working through receptor endocytosis to modulate the activation of FGFR/VEGFR/PDGFR and JAK/STAT signaling [C. elegans NDK-1 plays an important role in gonadal development [Xenopus, the NME4 homolog, NM23-X4, is essential for the development of the retina [Work with o adults . In the ignaling . In C. eelopment . In zebrelopment . In Xenoe retina . In micee retina ,177, bute retina . Furthere retina . In mouse retina . Moreovee retina ,181,182.e retina . Furtherawd and shibire (dynamin homolog) in the Drosophila demonstrates that awd functions as a GTP supplier to dynamin to mediate endocytosis [Besides development and cancer, there are studies showing the importance of NME in other biological contexts. Work with ocytosis . This haocytosis ,102. NMEocytosis ,184. Morocytosis , and NMEocytosis . All theThe detection of histidine phosphorylated NME has been challenging given that, until five years ago, we lacked the appropriate tools to selectively detect 1-pHis and 3-pHis in proteins. This is mainly why most of the work done so far has not explored NME protein-histidine kinase activity by evaluating changes in pHis protein levels. This began to change with the development of stable phosphoryltriazolylalanine (Tza) analogs of 1-pHis and 3-pHis by the Muir and Webb groups ,187, whiThe development of sequence-specific and sensitive monoclonal antibodies to individual pHis-containing proteins, and pH118 NME1/2 in particular, will be important for our understanding of the significance of NME protein histidine kinase activity in diverse cellular activities. There is a need for additional molecular tools in this field of study and complementary approaches, such as the development of selective NME histidine kinase inhibitors, would be valuable. Given the rapidly increasing amount of data and the identification of new histidine phosphorylated proteins, there is no doubt anymore that the extent of histidine phosphorylation was underestimated for many years but is now being recognized as an important regulatory post-translational modification. Many of the signaling roles of histidine phosphorylation remain to be explored, and important questions include whether other undefined histidine kinases and phosphatases exist and in what ways the addition of a phosphate to a histidine residue can initiate downstream signaling.