Epigenetic mechanisms finely regulate gene expression and represent potential therapeutic targets. Cambinol is a synthetic heterocyclic compound that inhibits class III histone deacetylases known as sirtuins (SIRTs). The acetylating action that results could be crucial in modulating cellular functions via epigenetic regulations. The main aim of this research was to investigate the effects of cambinol, and its underlying mechanisms, on cell differentiation by combining wet experiments with bioinformatics analyses and molecular docking simulations. Our in vitro study evidenced the ability of cambinol to induce the differentiation in MCF-7, NB4, and 3T3-L1 cell lines. Interestingly, focusing on the latter that accumulated cytoplasmic lipid droplets, the first promising results related to the action mechanisms of cambinol have shown the induction of cell cycle-related proteins (such as p16 and p27) and modulation of the expression of Rb protein and nuclear receptors related to cell differentiation. Moreover, we explored the inhibitory mechanism of cambinol on human SIRT1 and 2 performing in silico molecular simulations by protein–ligand docking. Cambinol, unlike from other sirtuin inhibitors, is able to better interact with the substrate binding site of SIRT1 than with the inhibition site. Additionally, for SIRT2, cambinol partially interacts with the substrate binding site, although the inhibition site is preferred. Overall, our findings suggest that cambinol might contribute to the development of an alternative to the existing epigenetic therapies that modulate SIRTs. These share a catalytic region of about 270 amino acids formed by a Zn-binding domain and a Rossmann fold hosting the NAD+ cofactor. SIRTs can combine deacetylation with the hydrolysis of NAD+ by forming 2′-O-acetyl-ADP-ribose and releasing nicotinamide (NAM), a feedback inhibitor of SIRTs. These enzymes exert their deacetylase activity on a variety of substrates: transcription factors, tumor suppressors (p53), and regulators of metabolism. It has been observed that SIRTs promote cell survival during stress through the deacetylation of histones and non-histone proteins that are essential to cell cycle regulation.The interest in the involvement of epigenetic alterations on the development of metabolic and oncological diseases is constantly growing . Epigened ageing . In the In mammals, SIRTs are also involved in cell differentiation and adipogenesis through interactions with the peroxisome proliferator-activated receptor-γ (PPARγ), a member of a class of nuclear hormone receptors regulating adipogenesis . The funThe role and functions of SIRTs in cancer development have not yet been fully elucidated, above all because they play different roles in different kinds of cancer. It is known that SIRT1 has an inhibitory effect on the p53 tumor suppressor and on other genes involved in the stress response, suggesting a pro-carcinogenic role of this SIRT. Furthermore, antisense oligonucleotides directed against SIRT1 induce apoptosis in lung cells, showing its therapeutic potential in lung cancer . SIRT2 c+-dependent HDACs belonging to class III (sirtuins) are not inhibited by conventional HDAC-I such as TSA and SAHA [+, nicotinamide non-selectively inhibits SIRTs through competition to the NAD+ binding site [Transcriptional dysregulations, related to numerous human diseases, are due to alterations in the equilibrium of HAT/HDAC that so constitute the potential target of new drugs such as the HDAC inhibitors (HDAC-I). NADand SAHA . Among tand SAHA ,16. Howeing site . The deving site  identifi50 values of 56 and 59 μM, respectively. Cambinol had weak inhibitory activity against SIRT5 (42% inhibition at 300 μM) and no activity against SIRT3. Class I and II HDACs are not sensitive to inhibition by cambinol, while SIRT4, SIRT6, and SIRT7 do not possess in vitro deacetylase activity. Enzymatic activity studies using NAD+ cofactor and acetyl-histone H4-peptide as substrate of sirtuins showed that cambinol is a competitive inhibitor toward H4-peptide, but not toward NAD+ [+ binding to sirtuins. This evidence suggests that it does not affect other NAD+-dependent enzymes, such as dehydrogenases, specifically blocking the sirtuin activity. It is this peculiarity that makes cambinol a drug candidate with potentially reduced adverse effects in the therapeutic use.Cambinol showed in vitro inhibitory activity of the human deacetylases SIRT1 and SIRT2, with ICard NAD+ . TherefoMore recently, new research is revealing the molecular interactions in the sirtuin–inhibitor complexes and the underlying mechanisms of how these inhibitors have an impact on cell growth and differentiation . Therefo2 atmosphere in formulated Dulbecco’s Modified Eagle’s Medium , completed with bovine calf serum to a final concentration of 10%, 1% L-glutamine, 1% ampicillin/streptomycin, and 0.1% gentamicin. Human acute promyelocytic leukemia NB4 cell line, provided by M. Lanotte , was grown at 37 °C in 5% CO2 atmosphere in RPMI-1640 medium , supplemented with 10% heat-inactivated bovine calf serum, 1% L-glutamine, 1% ampicillin/streptomycin, and 0.1% gentamicin. The cell lines were treated for 5 days (3T3-L1 and MCF-7) and 7 days (NB4) with 50 μM cambinol . Cell morphology was analyzed after staining by Oil Red O solution (Merck Life Science) for 3T3-L1 and MCF-7 cells, and after staining by May–Grünwald–Giemsa  for NB4 cells. Then, 3T3-L1 cells were treated with cambinol to a final concentration of 50 μM, with a drug cocktail consisting of troglitazone (5 µM), insulin (1 µg mL−1), and dexamethasone (1 µM), and their combination cocktail–cambinol for the time periods indicated in each experiment. After the treatments as above, 3T3-L1 cells were analyzed for adipogenic differentiation by Oil Red O staining following the protocol reported in Bontempo et al. (2015) [The 3T3-L1 mouse cell line  and MCF-. (2015) .5 cells/mL) and then collected after the treatments, centrifuged, and resuspended in PBS 1× containing propidium iodide , sodium citrate (0.1%), and NP40 (0.1%). Cell cycle was analyzed using the FACScalibur flow cytometer with ModFit technology (Becton Dickinson) and cell death levels were measured as pre-G1 DNA fragmentation.For cell cycle analysis and evaluation of pre-G1 phase, samples were processed and analyzed as previously reported by Bontempo et al. (2013) . Brieflyg for 30 min, protein concentration was determined, and protein extract aliquots of 40 µg were separated by 12% polyacrylamide gel electrophoresis and blotted as previously described [2, 1.5 mM NaMO4, 1 mM NaF) with protease inhibitors (Sigma), 1 mM DTT, and 0.2 mM PMSF. Briefly, 650 µg was pre-cleared with 20 μL A/G Plus Agarose . Mouse anti-PPARγ  or purified mouse IgG  antibodies were added and IP proceeded overnight at 4 °C. Then, 30 μL A/G Plus Agarose was added and incubated for 2 h. Finally, 20 μL of 2× concentrated electrophoresis sample buffer was added. Following Western blot analyses, mouse anti-PPARγ , mouse anti-acetyl-Lysine , and normal mouse anti-IgG  were used. Secondary antibodies  were used with a dilution of 1:10,000. At the end, the target proteins were visualized by chemiluminescence  and semi-quantified by densitometry using the Java-based image-processing and analysis software ImageJ .Cell lysis was carried out as previously reported . After cescribed . Western+ cofactor was present for SIRT1 and 2, while Carba-Nicotinamide-Adenine-Dinucleotide (CNA) was present for SIRT3. Since it is known from the literature that the binding to the inhibitor causes an important movement in the NAD+ molecule, blocking its kinked active conformation [+ for SIRT1 and 2 and of CNA for SIRT3. Water molecules present in the active site of SIRT1 and 2 crystals were kept in order to study their possible functional role in the inhibitor binding. Redocking with their respective inhibitors and exploiting blind and focused rigid body docking procedures were also performed, according to the procedure in use in our laboratory and similar studies from the literature [In order to analyze the interactions between cambinol and SIRTs, molecular docking studies were performed. Three-dimensional structures of SIRT1, 2, and 3 available in Protein Data Bank (PDB)  were anaormation , moleculterature . For betterature , proteinterature , and docterature . ParametThe images of molecular structures were generated using the software UCSF Chimera  or AutoDIn this study, the differentiating action of cambinol was initially evaluated with a preliminary morphological screening in the solid and hematological tumor cell lines MCF-7 and NB4, respectively. Cambinol-associated effects, whose antiproliferative and proapoptotic actions are already known in the tumor cell lines, were evaluated in comparison to the 3T3-L1 non-tumor cell model. Interestingly, we found in all three cell lines a differentiating effect after treatment with 50 μM cambinol A. MCF-7,−1 insulin, and 1 μM dexamethasone) whose differentiating effects have already been reported [Successively, 3T3-L1 cells were separately treated with 50 μM cambinol, cocktail of drugs , and a cocktail–cambinol combination. The analysis of the cell cycle showed that cambinol did not exhibit cytotoxic effects, as detected by the low percentage value of cell death in the pre-G1 phase  of the cell number in the G2-phase of the cell cycle after 1 day of treatment, associated with a reduction (22%) in the cell number in the G1-phase A. This tIn order to tentatively characterize from a molecular point of view the differentiation activity induced by cambinol under physiological-like conditions of 3T3-L1 cells, a model of adipocyte maturation, we analyzed the expression of various protein factors that can play a key role in the control of the cell cycle and in the induction of differentiation. We evaluated the expression of the regulatory cell cycle proteins p16 and p27, both known to cause cell cycle arrest through the inhibition of cyclin-dependent kinases (CDKs) ,34.Overall, as shown in Several nuclear receptors are involved in the cell differentiation process, including the retinoic acid receptors (RARs), which are ligand-controlled transcription factors functioning as heterodimers with retinoid X receptors (RXRs) to finely regulate cell growth, differentiation, survival, and death in different cell systems after activation by retinoic acid (Ra) binding . Once inAnother important member of the nuclear receptor superfamily of ligand-dependent transcription factors is PPARγ, whose role in adipose tissue differentiation has been extensively studied . The PPAThe retinoblastoma tumor-suppressor family proteins (pRb) are key factors in the control of cell proliferation and are also known as “pocket proteins” ; their o+-dependent deacetylases (class III HDAC or sirtuins) by specifically modulating the activity of the human SIRT1 and SIRT2 [It is well-known that cambinol acts as an inhibitor of NADnd SIRT2 . Therefond SIRT2 . PPARγ ind SIRT2 ,46,47. OSince the observed effects of cambinol are the result of sirtuin inhibition, it was interesting to explore the inhibitory mechanism of cambinol on human SIRT1, 2 and 3 in silico by protein–ligand docking simulations.+ shows a difference of about 1 Kcal/mol between the binding to the substrate site  and to the canonical inhibition pocket . Better values are obtained for the non-canonical site under other docking conditions . It is important to underline that the redocking analysis, performed exploiting the inhibitor co-crystallized with the protein structure selected, showed a better binding energy  for binding to the canonical inhibition cavity, but a worst binding energy for the site of the substrate access   . These r+ with functional water molecules display a higher propensity of cambinol to occupy the same position detected for the co-crystallized inhibitor, i.e., the inhibition site. This finding is in agreement with previous work that simulated an interaction between cambinol and SIRT2 [+, the binding energy value at the inhibition site is very similar . However, the position is slightly shifted toward the substrate binding site, with a partial occlusion of its access. This finding confirms the experimental results reported in the literature at the molecular interaction level [+, forming H-bonds with the cofactor and inhibiting its correct conformation , which is in agreement with the literature [In the case of SIRT2 and cambinol, the results of both docking approaches, i.e., whether flexible or rigid NADnd SIRT2 . The binon level , which iormation . Moreoveterature .+ and water molecules in the active site, docking in the presence of the cofactor but without water molecules, and docking without all heteroatoms) confirmed for SIRT1 the possibility of cambinol to bind to both the inhibition and substrate binding sites, and for SIRT2 the preference for the inhibition site .The docking simulations of the interaction of cambinol with SIRT3 show that, under blind conditions, neither the inhibition site nor the binding site are identified as energetically preferred, and the ligand is positioned on the protein surface without specific preferences. In particular, the binding site of the co-crystallized inhibitor was not recognized at all as a possible binding site for cambinol. Focused docking between SIRT3 and cambinol with NAD/mol see . When NABy comparing the results of the molecular docking of cambinol with the three SIRTs see , the besSince the docking results indicate a preference of cambinol for the substrate binding site over the inhibition site only in the case of SIRT1, we investigated the detail of the residues at the interface, as reported in the last column of Moreover, the comparison of the two sequences indicates a good level of conservation of the residues interacting with cambinol at the canonical inhibition sites , which iAltogether, the findings presented here highlight the ability of cambinol to induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes, likely by modulating important nuclear receptors. Furthermore, the results on the identification and characterization of the effects of cambinol suggest that the inhibition of SIRTs can be used as a treatment for human pathologies in which they perform essential functions. The identification of other SIRT targets involved in the pathogenesis of metabolic and tumor pathologies paves the way for the possibility of revealing other contexts where the inhibition of deacetylases represents a real opportunity for a therapy. The ability of cambinol to specifically inhibit some SIRTs makes this synthetic molecule a potential model structure in the research and development of new therapeutic agents that are able to decisively counteract various human pathologies such as tumors and metabolic alterations. Our molecular docking studies, showing that cambinol is potentially able to interfere with substrate binding, indicate possible differences in the mechanism of the inhibition of SIRT1 and SIRT2. However, further studies are necessary to fully understand the molecular mechanisms underpinning the cellular responses and SIRT inhibition for the potential applications of cambinol and its derivatives as candidate drugs.


There is significantrigidification of the structure involving rearrangement of the cTnI(1–33)–cTnCinteraction and changes in the distribution of the cTnC helix A/Bangle, troponin I (cTnI) switch peptide (149–164) docking,and the angle between the regulatory head and ITC arm domains. Thefamilial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits astructure inherently more rigid than the wild type, with phosphorylationreversing the direction of all metrics relative to the wild type.Adrenaline acts onβ1 receptors in the heart muscleto enhancecontractility, increase the heart rate, and increase the rate of relaxation(lusitropy) via activation of the cyclic AMP-dependent protein kinase,PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptideof cardiac troponin I is responsible for lusitropy. Mutations associatedwith cardiomyopathy suppress the phosphorylation-dependent change.Key parts of troponin responsible for this modulatory system are disorderedand cannot be resolved by conventional structural approaches. We performedall-atom molecular dynamics simulations (5 × 1.5  This is achieved by theaction of adrenaline on β1 receptors in heart muscle cells,leading to enhanced contractility and a faster heart rate. β1receptor activation leads to cAMP production. cAMP acts directly onmembrane channels and activates the cyclic AMP-dependent protein kinase,PKA. PKA phosphorylates a number of membrane and contractile proteinsincluding two serines (22 and 23) located in a 33-amino-acid N-terminalcardiac-specific extension to cardiac troponin I (cTnI).2 Phosphorylation of serines 22 and 23 causes a two- to threefolddecrease in myofilament Ca2+ sensitivity and a correspondingincrease in the Ca2+ dissociation rate that determinesthe relaxation rate.4 This small change is sufficientto allow the lusitropic response to adrenergic stimulation, sincethe faster relaxation rate of the cardiac muscle due to cTnI phosphorylationcontributes to shortening the cardiac muscle contraction–relaxationcycle, allowing for efficient contraction at a faster heart rate.Interaction between myosin filaments andactin-based thin filamentsin cardiac muscle generates a contractile force that causes the propulsionof blood around the circulatory system. Ca2+-sensitivity shifts caused byphosphorylation or interfering with them such that Ca2+ sensitivity becomes independent of phosphorylation level (uncoupling).5 A well-studied example of this is the mutation in cardiac troponinC (cTnC) G159D, which causes familial DCM6 and has also been shown to uncouple cTnI phosphorylation from modulationof Ca2+ sensitivity9  and familial dilated cardiomyopathy (DCM). Importantly, suchmutations commonly affect the PKA-dependent troponin modulatory system,either mimicking the Caitivity9 1A. Uncou15 can only partially describe muscleregulation. Ca2+ binding and switching on the thin filamentare multistep dynamic processes, but current methods only show staticstructures. More importantly, key parts of troponin involved in contractilemodulation, including the cardiac-specific N-terminal TnI extension, which contains the PKA phosphorylation sites,the interdomain peptide (138–147) of cTnI, and the C-terminal18 amino acids of cTnT are not resolved experimentally, implying thatthey are disordered.Recent high-resolution cryo-EM images of wholethin filaments at4.8 Å resolution combined with the fitting of structures fromX-ray diffraction at 2.6 Å resolution18 have been employed but they havetheir limitations since only peptide fragments of troponin can bestudied. Recently, computational molecular modeling and all-atom moleculardynamics (MD) simulations have become the method of choice for understandingthe phosphorylation-dependent modulation of the cardiac troponin.20To understand the mechanism of regulationby troponin I phosphorylationand the effects of mutations, methods that can describe the dynamicsof troponin such as NMR20 could not ascertain with statistical significance if phosphorylationchanged any of the intrasubunit interactions of cTnI. It seemed morelikely that phosphorylation and mutations primarily act by alteringthe dynamics of the troponin molecule in ways that have not been previouslyanalyzed.18Our initial study of wild-type troponin21 At the tertiarystructure level, we observed that the principal motion within troponinwas a hinge motion between the NcTnC domain and the “ITC domain”.To test the validity of the model, we correlated the changes calculatedby MD with the experimentally reported effects of phosphorylationand mutation. Energetics calculations indicate that phosphorylationstrengthens the TnI “switch” peptide-NcTnC interactionin the wild type. In addition, phosphorylation changes the mean TnChelix A/B angle and interdomain hinge angle distribution and restrictstheir range of motion. In contrast, simulation of the cTnC G159D mutationindicated that the dynamics were restricted compared with the wildtype when unphosphorylated and that they changed in the opposite directionon phosphorylation, consistent with the known ability of the mutationto modulate Ca2+ sensitivity and block its phosphorylation-dependentchange.To resolve these questions,we have analyzed a substantially largerdata set (5 × 1.5 μs for each system of bisphosphorylatedand unphosphorylated state in the wild type and the cTnC G159D mutant)in a larger box (140 Å cube). We have identified significantphosphorylation- and mutation-dependent changes in the interatom interactionsbetween the disordered segments of troponin and the troponin coreusing the Arpeggio routine.20The cTn model used for simulations in thisproject was based onthat constructed and extensively studied in the group.2+-activated state ofcTn was used for simulation,which was built upon the crystal structure of the core domain of humancTn published by Takeda et al.12 (PDB accessioncode 1J1D).The missing amino acids were added to the model using UCSF Chimera.22 The cardiac-specific N-terminal peptide, residues 1–33 (NcTnI),was included using the NMR structure published by Howarth et al.23 (PDB accession code 2JPW) in the unphosphorylated conformation.Finally, all mutations present in the crystal were changed back tothe human sequence , and a redundant Ala residue presentin the NcTnI NMR structure was removed. Our model contains all threeregions of cTn with the full troponin C subunit and the partial structureof troponin I (1–171) and troponin T (202–288) thatform the core domain of the cTn complex. The complete computationalmodel of the core domain of cTn in the unphosphorylated state hasa total of 419 residues and 6812 atoms.The CaThe phosphorylated systemsand G159D mutation were created fromthis model by modifying Ser22, Ser23 of the cTnI protein, and/or Gly159of cTnC by manually editing the residues. The computational modelwas prepared in two different phosphorylation wild-type apo states:uP (no phosphorylation) and SEP (cTnI S22/S23 phosphorylated withnet charge −2 each).24 and the CUDA-acceleratedPMEMD25 code in the SPFP precision mode.26 All parameter and topology files were preparedusing LEaP, which is part of AmberTools19 with AMBER ff14SB force-fieldlibrary for amino acids and phosaa10 parameter library for phosphoserines(SEP).28All simulations wereperformed usingAMBER18 software suite+ ions as counterions and then solvated by addingwater molecules (TIP3P water model29).To simulate physiological conditions, water molecules were replacedwith ions at random to achieve a concentration of around 0.15 M NaCl.Each system underwent a five-stage minimization, heating, and equilibrationprocess before production runs:1.–1 Å–2. The minimization algorithm was switched from steepestdescent to conjugate gradient after 500 steps.An initial minimization of the solventfor a maximum of 10,000 cycles and the protein atoms, which were restrainedwith a force constant of 10,000 kcal mol2.A second minimization of all atomsin the system for a maximum of 2500 steps and switching from steepestdescent to conjugate gradient after 1000 steps.3.–1 Å–2), exceptthe N-terminal of TnI (1–32). The temperature was increasedlinearly over 50 ps.A first-stage heating from 0 to 100K under an NVE ensemble with weak restraints on the protein complex was used to estimate the binding affinity betweensegments of cTn with the rest of the protein complex. MMPBSA utilizesmolecular mechanics, the implicit continuum solvent model, and thesolvent-accessible surface area to estimate the enthalpy of binding.34 The KDE method uses kernels to represent eachdata point and estimates the underlying probability density functionof the observed data. The default parameters and bandwidth selectionmethods were used for KDE plotting with Gaussian kernels.Distributiondata were plotted using KDE with Seaborn (version 0.9.0).21 The result was aggregated to the residue levelwhere the specific interaction type between residues was either presentor not for each frame. The interaction data was summarized for alltrajectories for each system to produce the interaction counts wherethe highest possible count is the total number of frames for eachsystem (37500).Arpeggio was used todetermine the atomistic interactions between all interacting atompairs based on atom types and positions, for all available frames.3–85;Head: the average of C-alpha coordinates of TnC94–157;Hinge: the averageof C-alpha coordinates of TnC241–251, TnI69–76End of IT arm: the average of C-alpha coordinates ofTnTHinge anglewas defined asthe angle between the regulatory domain (vector from hinge to head)and the IT arm (vector from hinge to end of the IT arm). The coordinatesof the three points were defined as following:36 The end points of thehelix vector were calculated by taking the average of the coordinatesof 10 alpha carbons from each end of the helix. A Python reimplementationof Interhlx  was used for the A–Bangle calculations.The interhelical angle was definedas the supplement of the angle between helix A (TnC 14–25)and helix B (TnC 38–47) of troponin C, similar to the conventionused in previous literature.For eachsystem , the pairwise root meansquare deviation (RMSD) was calculated for the five production runtrajectories based on all heavy atoms. The conformation that has thelowest combined distance to all other structures within the groupwas picked as the representative structure. For the illustrationsof interactions profiled by Arpeggio, the representative structurethat most closely resembled the described interactions was chosen.2+ in the bisphosphorylated(SEP) and unphosphorylated (uP) states for both wild-type troponinand the cTnC G159D mutant (G159D). We show results for the doublynegatively charged phosphoserine since this is the species likelyto be present at physiological pH. Five 1.5 μs runs were performedfor each condition, and structural and dynamic properties were analyzed.We performed extensive MD simulations of thefull troponin core(419 amino acids) in the presence of Ca37 Wefound that the variability of RMSD was essentially time independentand varied randomly between runs . This was also true for all the other metrics derived fromthe trajectories . We therefore combined data from all five runs in our analysis.We calculated andplotted pairwise root mean square deviation (RMSD2d) graphs for theindependent simulation trajectories.Supplement Figure 4). RMSF plots clearly identify the disordered segments of troponin,in particular the N-terminus of cTnI , the cTnIinterdomain peptide region (137–148), and the C-terminus oftroponin T (278–288)  with phosphorylation resultingin an approximate 33 to 50% reduction in RMSF values. The RMSFs forG159D, similarly, do not change significantly with phosphorylationand are not significantly different from the wild type.21 toquantify phosphorylation and mutation-related differences. Arpeggioidentifies and classifies interactions between atoms based on proximityand angles. We then summarized the counts for H-bond, ionic, aromatic,and van der Waals interactions between all residue pairs. Full detailsof this analysis are given in Supplementary Table 1 shows the numerical stability of theinteractions that change significantly with phosphorylation or mutationwith a detailed analysis, and complete results are in Supplementary Data Set 1. The interaction countsare illustrated in heat maps ,and representative structures are shown in We have used the ArpeggiopackageResidues 1–35 of NcTnI, including phosphorylatableserines22 and 23, are positioned over the NcTnC domain 3A–D. AddPhosphorylation significantly modulates cTnI–cTnC,cTnI–cTnI,and cTnI–cTnT interactions. In the unphosphorylated troponin,the major interaction between NcTnC and NcTnI is cTnC Asp33, in theEF hand loop I, with cTnI Arg19, 20, and 21 . The resultingshift of NcTnI–NcTnC interactions changes the location of NcTnIon NcTnC and results in Lys39 in the B helix being pulled toward thephosphorylated serines of NcTnI, confirmed by the formation of a hydrogenbond interaction between cTnC Thr38 and cTnI (+32%). As a consequence,there is also a very noticeable change in the orientation of the endof helix B relative to the A helix. There is a loss of interactionof NcTnC Glu40 with NcTnI Arg20/21 (−6%/–20%). Uniquely,there is a strong phosphorylation effect on the aromatic interactionof the phenylalanine residues in the A and D helices. While the interactionbetween the end of the A helix Phe24 with the start of the D-helixPhe74 and 77 is relatively constant, the very end of the A helix Phe27increases its interaction upon phosphorylation with the D-helix Phe77(+13%).At the same time, new intra-NcTnI interactions are formedbetweenthe phosphate groups of Ser22 and Ser23 and arginines in NcTnI 2B and 3DThe Arpeggio analysis reveals interactions between the C-terminusof cTnT and both cTnC and cTnI not previously noted 2C–E. TheThere are H-bond interactions between the CcTnT Lys280 and thephosphorylated Ser22 and Ser23 of NcTnI that are completely absentin the unphosphorylated state 2D. ThereThe troponin I switch peptide,149–164, is docked onto thehydrophobic patch of NcTnC formed by helices A and B. It is anchoredto NcTnC by a stable interaction between NcTnC Glu19 and cTnI Arg161,but the repositioning of cTnC helix B upon phosphorylation altersthe hydrophobic patch structure and results in its repositioning.There is a noticeable loss of interactions between cTnC helix B andthe C-terminal part of the switch peptide. The interaction of NcTnCLys43 with the switch peptide residues Glu164 (−16%) and Asp167decreases, and the interaction of cTnC Arg46 with Glu164 and Asp167also decreases. At the N-terminal end of the switch peptide, thereis around 12% increased hydrogen bonding between cTnC Cys84 and cTnIAla150 and cTnC Ser89 and Gly91 with cTnI Gly159 2F,G.Supplementary Figure 5.The N-terminaldomain of troponin C is linked to the ITC domainby two unstructured peptides that cross the interface: cTnC 86–95and cTnI 136–148. The configuration of the two peptides definesthe relative orientations of the two domains. Rearrangement of cTnChelix B on phosphorylation alters helices C and D. On phosphorylation,contact between the end of D-helix Arg83 and residues Asp88 and Glu155is weakened, with the linker moving away from the D helix. At thesame time, the linker region, Asp88 and Gly91, moves collectivelytoward CcTnC, accompanied by changes in the E helix. At the startof the E helix, Ser93 is moving away from the rest of the helix. Thisis reflected in the reduction of its interactions with Glu95, Glu96,and Leu97 .The overall effect is that the cTnC linker peptide lengthens uponphosphorylation increasing the distance between the NcTnC and CcTnCdomains 3E.The iRepositioning of the cTnI switchpeptide relativeto cTnC helicesA and B on phosphorylation alters the adjacent cTnI interdomain peptide,136–148. The cTnC C helix moves with the B helix, thus alteringits interactions with the middle of the cTnI interdomain peptide.The arginines in this peptide  form ionicinteractions with cTnC, notably at the start of helix C and the endof helix D that shift on phosphorylation. Overall, upon phosphorylation,the cTnI interdomain peptide repositions toward the start of C helix3F.Supplement Figure 6G).Examiningfirst the effects of the G159D mutation on cTnC, whichare phosphorylation independent, the most significant change fromthe wild type is the formation of a strong ionic and hydrogen bondinteraction between Arg83, at the bottom of the D helix, and Asp159,at the end of the H helix (∼80% of the simulations). Arg83also forms a strong ionic interaction with the N helix of cTnC, whichis phosphorylation dependent in the wild type . The key phosphorylation-dependent interactionsbetween cTnI Arg19, Arg21, Ser22, Ser23, and Arg26 with cTnC Lys33and Lys39 change similar to the wild type. However, reversal of theobserved changes in interactions upon phosphorylation for G159D relativeto the wild type is frequent in the region of the interdomain interface.Thus, the ionic interaction of cTnC Asp25 and Glu32 with cTnI Arg12in the G159D phosphorylated state decreases from the unphosphorylatedstate (−14%) while in the wild type they increase (+7%). Thisis particularly evident in the interaction of the linker region ofcTnC with itself; for residues Lys90 with Glu94, the ionic interactionsincrease upon phosphorylation (+3%), while for the wild type, theydecrease (−6%). The collective effect in the linker regionof G159D is that phosphorylation induces a shortening of the linker,which is the direct opposite of the observation in the wild type . This reversal of behavior upon phosphorylationof G159D relative to the wild type is also observed in the interactionsof the EF3 hand of TnC with CcTnT (Supplement Table 1).Structural differences accompanying phosphorylationof G159D, in both magnitude and sign, can be observed with respectto the interaction of the N helix of cTnC and the beginning of theH1 helix of NcTnI  has previouslybeen identified as a suitable metric for evaluating the effect ofCa36 For the unphosphorylated wild type, thisis 18%, and for G159D, it is 1%; the corresponding values for thephosphorylated states are 1% (wild type) and 7% (G159D). The A/B angleof highest density using KDE, however, indicates that while thereis a small difference between wild-type unphosphorylated and phosphorylatedin the same direction as the mean angle value, there is only the smallestof differences between the phosphorylated wild type and G159D in bothstates. The FWHM parameter (arameter 1 demonst147–163), which is in close contact with theA/B angle (hydrophobic patch) region of NcTnC; cTnI34–71, which interacts with the C-terminal domain of cTnC and is closeto the G159D mutation site; and finally, the cTnI interdomain peptide(TnI131–147), which crosses from the NcTnC domainto the ITC domain and is hypothesized to be critical in the contraction/relaxationprocess. The distribution of MMPBSA values is shown in Supplementary Figure 7.The interaction energy between regions of cTnCand cTnI has beenevaluated using the MMPBSA methodology 1. Three G values for the wild type and G159D mutant. This resultcorrectly reproduced the trend found by the wild-type NMR titration;comparing the difference in ΔG upon phosphorylationfor the wild type, there is, perhaps fortuitously, a similarity inthe magnitude between experiment and simulation, ΔΔG = −1.2 and −3.5 kcal/mol, respectively.38We found that theswitch peptide binds to the hydrophobic patchmore strongly upon phosphorylation, with similar ΔΔG = −3.9 kcal/mol); conversely, there was decreasedaffinity in the TnC G159D mutant .The cTnI interdomain peptide binding affinityof the wild typewas observed to increase on phosphorylation , in agreement with previous measurements.39 In the phosphorylated state, wild-type and G159D mutantcTnC both showed a higher affinity for the cTnI34–71 region than in the unphosphorylated state, being more marked inthe wild type.Troponin C is anchored in the complex throughan interaction ofthe C-terminal domain with TnId to assess the effect size between the means of the data sets whenmaking comparisons (Supplementary Table 2). Low values often correlate with bimodal distributionsMD simulations produce a large number of correlatedsamples forevery metric. The distributions are often non-Gaussian; therefore,traditional statistical tests are not appropriate. Effect size isa more descriptive metric in that it qualitatively describes how largethe difference is between the two samples. Traditional statisticaltests tend to deal with experimental data with a low number of observations.This means that most of the comparisons stemming from this work arelikely to produce significant test statistics when the null hypothesisis that the mean of the two distributions is different although theactual difference could be negligible. Effect size is a more descriptivemetric in that it qualitatively describes how big the difference isbetween the two samples. Here, we used Cohen’s 2+ sensitivity andincreases the rate of Ca2+ release from troponin C. Itis a major determinant of the lusitropic response to adrenergic stimulation40 The molecular mechanism of this process has remained unknown longafter the mechanism of the troponin Ca2+ switch itselfwas defined.41 Investigation of this subtle process at the atomic level poses achallenge for structural biology since the change in Ca2+sensitivity is only about twofold,42 andkey parts of the troponin modulation and regulation system are disorderedand cannot be fully resolved by most methodologies. In this study,we have used MD simulations of the cardiac troponin core to understandthe molecular mechanism by which phosphorylation of serines 22 and23 and a DCM-linked mutation, cTnC G159D, modulate Ca2+ regulation in cardiac muscle.The phosphorylation of serines 22 and 23of troponin I in cardiactroponin modulates the myofilbrillar Ca43 Our extended simulations using a 140 Å cubic water box hasavoided these problems and now allows us to demonstrate that Ca2+-saturated troponin exhibits a considerable range of motionand that NcTnI, the cTnI interdomain peptide, and the CcTnT can fluctuateover a range of conformations. The N-terminal peptide of troponinI is located on the surface of the N-terminal domain of troponin C.Phosphorylation and mutations alter the dynamics of troponin, andby using the Arpeggio technique, we have been able to define significantlocal changes in the populations of these conformations that couldexplain the action of phosphorylation and mutations.In previous studies of troponinusing MD methods, conclusions weredifficult to sustain due to questions about the box size used andthe length of simulations.The direct effectof phosphorylation at serines 22 and 23 is alocal rearrangement in which basic amino acids, including neighboringarginines in NcTnI  and cTnC lysine 39 tend toform a cluster of ionic bonds with the phosphate groups of phosphoserines22 and 23 at the expense of interactions between cTnI and cTnC, particularlythe cTnC Asp33 interaction with Arg19, 20, and 21 of NcTnI. The pullingof cTnC Lys39 toward cTnI serines 22 and 23 impinges upon helix Band its orientation relative to helix A and the hydrophobic patchthat the switch peptide binds to, manifested as the change in interhelicalangle and increased affinity of the switch peptide for cTnC. Longer-rangeallosteric consequences of the helix B rearrangement include a repositioningof the switch peptide and changes in the peptides crossing the interdomaininterface that can account for the observed changes in hinge angle.There is considerable rearrangement and lengthening of the interdomain“linker” region of cTnC, residues 87–97, uponphosphorylation. This has an effect on the disposition of the NcTnCdomain relative to that of the CcTnC domain, with the two domainsbecoming closer. There are also changes in the interaction of theCcTnT and the EF3 and 4 hands of CcTnC resulting in the close couplingof CcTnT and CcTnC.Finally, there is also a strong local changein the positioningof the cTnI interdomain peptide (cTnI residues 139–148) relativeto the C and E helices of cTnC. Upon phosphorylation, the cTnI interdomainpeptide moves to position itself closer to the start of the cTnC Chelix, losing its interaction with the CcTnC E helix.2+ switching, as summarized by Stevens et al.44 Ca2+ activation of troponin is a two-step process: first,Ca2+ binds to site II of troponin C, and then the hydrophobicpatch of cTnC opens  andthe switch peptide binds to the open patch forming the active state.9 Since our studies are all in the presence of Ca2+ bound,we only see the active (“open”) state.The local changes in troponin describedabove lead to macroscopic changes that can be compared with experiment.We have based our analysis on the consensus model of Ca13 The hinge angle is altered by Ca2+ activation45 and is also proposed to be modulated by phosphorylation46 The simulations indicate thatupon phosphorylation, the distribution of hinge angles in wild-typetroponin shows an increase in the mean angle with a significant rigidificationand decrease of the accessible range. This is consistent with thestudies of Hwang et al.,17 who proposedthat phosphorylation modified the positioning of NcTnC relative tothe ITC domain, based on NMR measurements. It is notable that theC terminus of troponin T (CcTnT), previously not included in MD simulations,19 is located within the interdomain interfacewhere its interactions with NcTnI and cTnC are phosphorylation dependent,thus possibly accounting for the known role of this peptide in cTnregulation48The principalmotion of troponin is a hinge-like motion between the two quasi-rigiddomains of troponin, NcTnC and the ITC domain.Using the A/B helix angleas our metric for the opening and closingof the hydrophobic patch, we found that, indeed, phosphorylation hasa significant effect on the distribution of interhelix angles withan increase from 82 to 99% distribution below 110°, which wasproposed as the threshold of the “open” state. Accompanyingthe hinge angle change, there is a significant rigidification anddecrease in the accessible range upon phosphorylation.38Finally,we note that interaction energy calculations from MD simulationsindicate that phosphorylation strengthens the interaction betweenthe cTnC hydrophobic patch and the cTnI switch peptide in quantitativeagreement with Baryshnikova et al. using NMR titrations.G switch peptide. MD shows us that phosphorylation uniformlyreduces the accessible range of whichever metric we observe, suggestingthat phosphorylation results in rigidification of the overall structureof the troponin complex.In summary, the global effect of phosphorylationon the dynamicsof the wild type is comparable with experimental measurements of changesin the interdomain hinge angle, A/B helix angle, and ΔΔ2+ sensitivity due to PKA phosphorylation of serines 22 and 23 thatis crucial for the lusitropic effect,8 and this maybe sufficient to cause the DCM phenotype.11 We found that the G159D mutation has significant effects upon thedynamics of troponin.To test further the correlation betweenexperimental data and thechanges in troponin dynamics due to phosphorylation, we studied thecTnC G159D mutation, which is associated with familial DCM. The keyeffect of the mutation is that it abolishes the shift in CaThe primary difference due to the G159Dmutation in cTnC is the formation of strong and phosphorylation-independentbonds across the bottom of the interdomain interface, particularlybetween cTnC Arg83 and Asp159; consequently, the range of hinge anglesis reduced. This affects the location of the apparent fulcrum of the“hinge” so that the phosphorylation-dependent changesin the peptides crossing the interdomain interface can result in oppositechanges consistent with the functional abnormalities caused by themutation.Unphosphorylated G159D troponin has a lower mean A/Bhelix anglethan the unphosphorylated wild-type troponin and a higher probabilityof being in the range of angles defined as the open state. The meanhinge angle is similar to that of the phosphorylated wild-type troponin,but the distribution is narrower. In general, unphosphorylated G159Dresembles the phosphorylated wild type more than the unphosphorylatedwild type.2+ sensitivitydue to PKA phosphorylation of Ser22 and 23.8 Itis noticeable that the interactions between NcTnI and NcTnC involvingcTnC Lys 33 or Lys39 and TnI Ser 22 and 23 and the arginines 19, 20,21, and 26 are the same as the wild type and change in the same wayon phosphorylation, independent of the mutation-related changes inthe interdomain region. This indicates conformational uncoupling thatmirrors the functional uncoupling caused by the mutation.Interestingly, the effect of phosphorylation on thesemetrics isin the opposite direction of the wild type. For instance, for theA/B metric, phosphorylation of G159D increased the mean angle by 1.9°and decreased the proportion below 110° (open state threshold)from 99 to 93%. Phosphorylation of G159D also shifts the interdomainangle distribution toward a lower mean angle and increased RMSF acrossthe whole system, particularly troponin C, indicating a global aswell as the specific effect of the point mutation. The findings thatthe wild-type response to phosphorylation, observed in our MD simulations,is disrupted by the G159D mutation correlates with the observationthat the G159D mutation abolishes the shift in Ca2+ activationpathway of troponin, consistent with an enhanced probability of theopen state on phosphorylation. Phosphorylation results in rigidificationof the overall structure of the troponin complex. We suggest thatthis stabilizes cTnI switch peptide docking to the hydrophobic patchof troponin C in the Ca2+-bound (open) state, and thishas an effect on Ca2+ sensitivity and Ca2+ release.The observation that an uncoupling mutation induces an opposite changein these metrics on phosphorylation supports this proposition. Thiscould be further tested with other uncoupling mutations or small moleculesthat can reverse uncoupling;49 it is noteworthythat most of the mutations in the troponin core that are reportedto uncouple are at the interdomain interface . Rigidification of the overall structureof troponin by mutations, mimicking phosphorylation, may explain howmany different mutations in various components of the thin filamentcan uncouple50The comparative results support our proposalthat the MD simulationsfaithfully report relevant changes in dynamics induced by phosphorylationand mutation of the troponin core. We observed limited phosphorylation-dependentchanges in the equilibria between a range of conformations of thetroponin core similar to those known to be on the CaIn vivo troponin is incorporated into the thinfilament and interacts with tropomyosin and actin. The troponin corehas been docked into recent high-resolution cryo-em images of thethin filament at both relaxed and activating Ca2+ levels,and the positions of the cTnI N terminus and cTnT C terminus, notincluded in the troponin core, have been resolved. Nevertheless, cryo-emstill does not provide any information about the disordered segmentsof troponin described here. An attempt has been made to model theNcTnC peptide into the em envelope and suggests there may be additionalinteractions with tropomyosin.51 With thenew thin-filament structural models as a starting point and with ever-increasingcapability of processors for MD calculations, an all-atom simulationof the thin filament, including the disordered regions, has becomefeasible.


The differential scanning calorimetry (DSC) results indicated that recombined strain B9-INP was an effective ice nucleator for controlling the supercooling point of distilled water at low concentrations. In the presence of B9-INP cells, model food systems, including sucrose solution and sodium chloride solution, different pH solutions froze at a relatively high subzero temperature, thus increasing the supercooling point by 5.8~16.7 °C. Moreover, B9-INP also facilitated model and real food systems to freeze at −6 °C. This recombinant strain not only improved the freezing temperature of food systems but also shortened the total freezing time, thus saving energy and reducing consumption. The results suggest that B9-INP has great application potential in the frozen food industry.To produce food-grade ice nucleators, a 3.77 kb ice nucleation gene ( Xanthomonas, Pantoea agglomerans (formerly Erwinia herbicola), and Pseudomonas. Previous studies have indicated that a beta-helix structure within the central repeat domain of INPs plays a critical role in regulating ice nucleation  cells were selected as the recipient bacteria for DNA manipulations. DH5α was routinely grown in an LB broth  with 20 µg/mL tetracycline. Pantoea agglomerans (acquired from the American Type Culture Collection (ATCC Cat. No. 27155)) was used as the parent strain for the iceE gene cloning. Bacillus amyloliquefaciens (BAX-9) was used for transformation experiments. BAX-9 harboring plasmid pHY300PLK (B9-INP) was grown in an LB broth. Plasmid pHY300PLK was the shuttle plasmid of E. coli and Bacillus amyloliquefaciens. The chromosomal DNA of Bacillus subtilis 168 (B. s 168) and Bacillus licheniformis (WX-02) was used as the promotor and terminator template, respectively.B. s 168 chromosomal DNA using primers sP43-F-BamHI and sP43-INP-R. The Tamyl terminator was amplified from WX-02 chromosomal DNA using primers sT-INP-F and sT-R-XbaI. A 3777 bp ice nucleation gene (iceE) was cloned from the total genomic DNA of Pantoea agglomerans using primers sINP-P43-F and sINP-T-R. The linearization of pHY300PLK vector was performed by BamHI and XbaI restriction enzyme double digestion. Then, the three PCR fragments were gel purified and ligated by the one-step cloning kit [E. coli DH5α. Tetracycline-resistant positive transformants were further verified by colony-PCR and sequencing. Recombinant plasmid pHY300-INP was electrotransformed into BAX-9.The P43 promoter was amplified from ning kit , and rec8 CFU/mL. BAX-9 was used as a control. The culture broth was centrifuged at 6000 r/min for 15 min, and bacteria were collected and dissolved in distilled water to prepare bacterial suspension. The resultant bacterial suspension was freeze-dried for ice nucleation activity assays and subsequent experiments.B9-INP containing the recombination plasmids pHY300-INP was grown in an LB broth at 37 °C with shaking at 200 rpm until a density of 10−5 mg/mL. Briefly, a 10 μL bacterium solution sample was sealed in a standard aluminum pan, with an empty pan as the control. The DSC unit’s temperature was decreased from 4 °C to −25 °C at a rate of 1 °C/min. The temperature point corresponding to the highest heat flow observed is referred to as the supercooling point. The distilled water containing 10 mg/mL BAX-9 was used as the control. The experiments were performed in triplicates [The determination of the supercooling point of bacterium solutions with different concentrations was conducted using a 200F3 differential scanning calorimeter  powered by liquid nitrogen and compressed nitrogen gas. The B9-INP was serially (10-fold) diluted in distilled water from 10 mg/mL to 10plicates .Sucrose solution and sodium chloride solution at different concentrations  and acid-based solution (pH 2~11) were prepared. The freeze-dried powder of B9-INP was added into each of the above 3 solutions to reach a final concentration of 1 mg/mL (according to the results of Different concentrations (m/m) of sucrose solution (10 mL) and sodium chloride solution (10 mL) containing B9-INP at different concentrations utilized in this study were subjected to radial freezing by placing them in plastic centrifuge tubes immersed in a stationary cooling bath comprising 50% ethylene glycol. The centrifuge tube was inserted into the float board so that the liquid level of the sample was at the same level as the bottom surface of the float board. The thermometer probe was inserted into the centrifuge tube with one end of the probe in the middle of the sample solution during the measurement. Three temperature gradients were set as −6 °C, −8 °C, and −10 °C, and temperature changes were recorded until the solution temperature remained stable .According to the above results, a constant temperature was set. The freezing temperature curves of orange juice, apple juice, egg white, whole egg, whole milk, and skimmed milk (each 10 mL) containing different B9-INP concentrations were plotted .−2 mg/mL), and 20 μL B9-INP (1 and 10−2 mg/mL) samples were, respectively, placed on microscope slides (at −18 °C). The ice crystal structure was evaluated utilizing a 10× lens (0.25 N.A.) microscope  equipped with a temperature-controlled system . The ice crystal structure images at the ice surface layer were captured [The 20 μL Distilled water, 20 μL BAX-9 (1 and 10captured .PCR primers were designed using Primer Premier 5 (5.00) software. The experiments were conducted with three replicates. The data were expressed as the mean ± SD of three replicates. The diagrams were plotted using Origin 2021 (9.8.0.200) software. SPSS Statistics 26 (26.0.0.0) was used for data processing and statistical analysis.iceE gene, and Tamyl terminator were cloned using primers via PCR. Recombination was verified using PCR colonies. As shown in The P43 promoter, −5 mg/mL. Previous research has indicated that the INP concentration is not linearly correlated with the supercooling point due to the impact of protein aggregation on ice nucleation activity [E. coli to the recent software simulations, the formation of dimers and higher-order oligomers has been reported to be indispensable for INPs’ activities [iceE gene in the Gram-positive bacterium B9-INP. Our results suggested that B9-INP exhibited significant efficacy as an ice nucleator in regulating the supercooling point of distilled water, even when present in low concentrations. In recombinant hosts, the threshold temperature for ice nucleation was usually lower than that in wild-type bacteria. The ice nucleation activity of B9-INP was only slightly lower than that of E. coli. One possible explanation is that Gram-positive bacteria have significantly different lipid compositions of the membranes from those of E. coli [Bacillus amyloliquefaciens, this issue can be circumvented.The effect of B9-INP on the supercooling point of distilled water was investigated using DSC A. The suactivity . From thtivities ,29,30,31tivities . However E. coli . FurtherThe increase in nucleation temperature contributes to saving a great deal of energy during the freezing processes. Given the correlation between the bacterium B9-INP concentration and its ice nucleation effect, 1 mg/mL B9-INP was selected in subsequent experiments.The impact of B9-INP on the supercooling point of sodium chloride solutions at various concentrations was explored through DSC A. The fiP. syringae; −2.5 °C for P. ananatis), B9-INP could still significantly increase the nucleation temperature of the model solution. Therefore, the addition of B9-INP can guarantee the freezing of some liquid foods at relatively high temperatures.Compared to that of the control, the supercooling point of distilled water added with 1 mg/mL B9-INP at different pH values was increased by 9.1~15.9 °C. Generally, adding any solute into a solution, whether it is organic or inorganic, will cause a decrease in its freezing point, preventing it from freezing. The nucleation of ice marks the time point when bulk water begins to freeze. Such a transformation might be attributed to the fact that INPs can efficiently promote ice formation by generating ice nucleators containing repetitive amino acid sequences. Previous studies of model foods have shown that with sucrose concentration increased, either by the addition of ice-nucleated bacteria or extracellular ice nucleators, the supercooling point decreased ,14. HoweThe effects of B9-INP on the supercooling point of solutions with different pH values were investigated C. The reThe impact of B9-INP on the freezing process of sodium chloride solution and sucrose solution was investigated A. As shoIn the sucrose solution group B, the frMany industrial processes rely on efficient freezing techniques, such as freeze storage, freeze drying, and freeze concentration. By utilizing subzero temperatures, it is possible to reduce freezing energy, increase productivity, and enhance the quality of various products ,23. ThisSome real foods were selected to test the ice nucleation activity of B9-INP. According to the results of the experiment in In the juice group B, orangeIn addition, the total freezing time of apple juice was 2594 s at the 1 mg/mL B9-INP and 3557 s at the 0.5 mg/mL B9-INP. The increase in the concentration of B9-INP saved nearly 27 percent of freezing time. The addition of 0.1 mg/mL of B9-INP could freeze whole egg liquid at −6 °C, but to freeze raw egg white required 1 mg/mL B9-INP, which might be due to the presence of the phospholipid in egg yolks. The ice nucleation activity genes have been successfully cloned and expressed in bacteria, zymomonas, and yeast so far . Our recThe ice crystals of distilled water, BAX-9 suspension, and B9-INP suspension were characterized. The addition of B9-INP cells increased ice nucleation temperature, altered ice formation patterns, and produced a special ice crystal texture . Fine paIn the food freeze concentration industry, a dry layer morphology greatly influences the heat transfer and ice sublimation rates during the primary drying process. Ice crystals formed during freezing determine the structure of porous layers . In previceE gene in Bacillus amyloliquefaciens. In the presence of B9-INP cells, model food systems, including sucrose and sodium chloride solutions, froze at a relatively high subzero temperature. B9-INP increased the supercooling point by 5.8~16.7 °C. In addition, B9-INP could also cause model and real food systems to freeze easily at −6 °C. This recombinant strain can not only increase the freezing temperature of the food system but also shorten the total freezing time, thus saving energy and reducing energy consumption. Our findings provide experimental data and theoretical support for the application of INPs in the frozen food industry.Bacterial ice nucleators exhibit great potential in the food processing industry. However, the application of bacterial ice nucleators is restricted in the food industry due to safety concerns over natural INP-expressing bacteria. To solve this problem, GRAS host cells can be adopted. In this study, we successfully expressed the


Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpros) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by coexpression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two noncanonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER-resident proteins, including at sites that could escape analyses based on the established consensus sequence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the COVID-19 pandemic. Nonstructural protein 3 (Nsp3), the largest nonstructural coronavirus (CoV) protein, is synthesized as a polyprotein with other Nsps. Nsp3s include domains that have a variety of functions related to viral pathogenesis and have two transmembrane domains that localize them to the endoplasmic reticulum (ER) when expressed alone or during the initial phases of infection. Topologically, all but a short region that binds Nsp4 faces the cytosol. Nsp3 and Nsp4, together with Nsp6, are required to form the double membrane ER-derived SARS-CoV replication/transcription complex ((P4)X(P3)G(P2)G(P1), using the numbering system of Schechter and Berger -CoV, include a single papain-like protease (PLpro) composed of a ubiquitin-like domain and protease domain. PLpro, which is situated N terminal to the two Nsp3 transmembrane domains, plays an essential role in the cleavage of CoV polyproteins into their constituent mature proteins, releasing Nsp1, Nsp2, and Nsp3 itself . For MERd Berger , where cnd at P4 . In addind at P4 , 9, 10. nd at P4 , 13, 14,nd at P4 , 17, 18.nd at P4 .versus transmembrane forms of the same PLpro would impact requirements for PLpro DUB activity and what the overall importance of membrane tethering is to immune suppression by SARS-CoV-2 PLpro. Nevertheless, there is substantial data to suggest that catalytic activity is dispensable for PLpro to oppose activation of multiple signaling molecules integral to the innate immune response.For SARS-CoV-1 and SARS-CoV-2, studies on ubiquitination have focused on antagonistic roles that PLpro might play as a deubiquitinating enzyme (DUB) toward activators of innate immunity mediated by a type 1 interferon (IFN) response. Here, ubiquitination of multiple proteins plays critical roles in activating signaling through K63 and other polyubiquitin linkages, rather than by targeting these proteins for degradation. In several studies examining either transmembrane or soluble cytoplasmic SARS-CoV-1 PLpro, attenuation of the IFN response was found to be independent of PLpro catalytic activity and attributed to disruption of protein–protein interactions , 22, 23.In addition to roles in the IFN response, the ER is a hub for other signaling, including cellular stress responses and pathways leading to cholesterol and fatty acid biosynthesis. Proteins in these pathways and others are regulated in part by a ubiquitin and proteasome-dependent homeostatic process known as ER-associated degradation (ERAD), which is also central to ER quality control , 28, 29.Despite the ER localization of Nsp3, there has been little investigation into a possible role for PLpro in modulating ERAD. We therefore examined the effects of transmembrane forms of SARS-CoV-2 PLpro on exogenously expressed INSIG-1, SREBP-1, and SREBP-2. Our findings reveal that catalytically active transmembrane PLpro impacts these proteins in different ways by decreasing ubiquitination and, unexpectedly, by endoproteolytic cleavage including at noncanonical sites. In contrast to the type I IFN response, all the effects we observe require the catalytic activity of PLpro and most require ER membrane anchoring of the enzyme. Overall, our observations suggest that PLpro potentially plays an underappreciated important role in determining the fate of ER proteins and proteins that partition into the replication/transcription complex during SARS-CoV-2 infection.A -schematic). Both truncations begin at the PLpro. One terminates immediately after the PLpro domain (Nsp3PLpro), while the other extends through the two predicted transmembrane domains and an adjacent amphipathic helix (Nsp3TM) (B and C).Full-length Nsp3 from coronaviruses are known to localize to the ER prior to formation of the viral replication/transcription complex, which itself incorporates ER membrane and ER proteins , 3, 4. TFig. 1D). Nsp3TM also results in increased levels of INSIG-1, while the cytosolic Nsp3PLpro does not (E). Similarly, unlike Nsp3TM, catalytically inactive Nsp3TM in which Cys111 of PLpro is mutated to Ala (Nsp3TMC111A) does not increase INSIG-1 levels in HEK293T cells (F), while both the WT and mutant forms of Nsp3TM coimmunoprecipitate INSIG-1 (A). Similar PLpro activity–dependent INSIG-1 stabilization is observed in HeLa cells (B). In contrast to INSIG-1, the level of the Null Hong Kong (NHK) variant of alpha1-antitrypsin, a luminal ER protein that is extensively employed as a model ERAD substrate (G) Importantly, active Nsp3TM but not Nsp3TMC111A results in a marked decrease in INSIG-1 ubiquitination without altering total cellular ubiquitination, as assessed after proteasome inhibition to prevent INSIG-1 degradation (H). These findings are consistent with transmembrane PLpro functioning as a DUB for INSIG-1.We asked whether Nsp3 affects levels of transfected INSIG-1, which is a multitransmembrane ERAD substrate. Nsp3FL markedly increases the level of exogenously expressed INSIG-1 and delays its loss during a cycloheximide (CHX) chase in HEK293T cells D. Nsp3TMdoes not E. Simila3T cells F, while  INSIG-1 A. SimilaLa cells B. In conubstrate , 43, 44,ubstrate G Importaradation H. These A). We then examined the effects of coexpression of different forms of Nsp3 on SREBP stability in HEK293T cells. When SREBP-1 is expressed with either Nsp3FL or Nsp3TM, but not with inactive Nsp3TMC111A, we observe an unexpected acceleration in the loss of SREBP-1 over 2 h of treatment with CHX . Overexpression of SREBP-1 also results in variable amounts of T7 immunoreactive species in the 70 kDa range . These have been observed by others . No evidence for these ∼45 kDa species, or of accelerated loss of full-length SREBP-1, is apparent with soluble cytoplasmic Nsp3PLpro . As full-length SREBP-1 in B and C is epitope-tagged with a C-terminal E-tag, we assessed a form lacking this tag and found it similarly affected by Nsp3TM (A). Except where indicated, E-tagged SREBP-1 is used in the rest of this study. Nsp3TM coexpression had no effect on the soluble NTD of SREBP-1, which corresponds to the active nuclear transcription factor (D and B). In striking contrast to SREBP-1, both Nsp3FL and catalytically active Nsp3TM increase the level and inhibit the degradation of SREBP-2 . This increase is not observed with Nsp3TMC111A. Soluble Nsp3PLpro also results in some increase in the level of SREBP-2, albeit to a lesser extent than transmembrane Nsp3TM .We next evaluated full-length ER-localized SREBP-1 and SREBP-2. Like Nsp3, SREBPs have two closely spaced transmembrane domains, with both their N and C termini facing the cytosol. Using N-terminal T7-tagged forms, we established that exogenously expressed SREBP-1 and SREBP-2 both colocalize with Nsp3 A. We they others  and may y Nsp3TM A. Exceptn factor  Figs. 2A. We theor D and B. In str4 position (LXGG) . Both with and without phosphatase treatment, the 318 truncation closely approximates the migration of the Nsp3-dependent cleavage products . Notably, this truncation lacks the basic helix-loop-helix leucine zipper motif, which is required for the transcriptional activity of the NTD . Additionally, in SARS-CoV polyproteins, a Leu is invariably found in the Pn (LXGG) . There a the NTD , 31, 46.A – site 1 and mutations). Neither of these eliminated the Nsp3-dependent species . We therefore entertained the possibility of more than one cleavage site in this region of SREBP-1, but the closest diGly are over 100 amino acids in either direction at aa 200-201 and the aforementioned aa 437-438. However, there is a AlaGly 23 amino acids from aa 318 at residues 340-341, which includes a Leu in the predicted P4 position . A deletion of amino acids 338-348 (Δ338-348) in combination with the A317-318 mutation resulted in loss of the lower molecular weight species . Importantly, Δ338-348 does not itself result in loss of these forms . These findings support two sites of cleavage in proximity. This is further supported by comparing the relative migration of these N terminal–containing species in the Δ338-348 deletion and the A317-318 mutation , each of which would leave only one of the two sites intact.To further evaluate this cleavage, we either mutated the diGly at 317 and 318 to Ala (A317-318) or deleted these amino acids (Δ317-318) . This demonstrates that the predicted cleavage site Gly (P1) is required for cutting, as is the Leu in P4, while the Arg at P5 is not . These findings implicate this region as a site of cleavage but suggest adequate flexibility to allow for Ala in P2. To determine whether the amino acid in the predicted P2 position is of significance, the Ala was mutated to either Leu, a larger hydrophobic amino acid, or to Glu, an acidic residue. Both resulted in a loss of cutting . Thus, site 2 represents a noncanonical Nsp3 cleavage site. Further confirmation of two sites is shown by comigration of cleavage products of SREBP-1 having only site 1 or site 2 intact with corresponding truncations at each of these sites (A). Similar results are observed in HeLa cells (B).The requirements for cleavage in the region encompassing Gly341, hereafter designated site 2, was examined by point mutation of individual residues to Ala in the context of SREBP-1 that also includes the A317-318 mutation, in what we designate as site 1. We compared these point mutations to SREBP-1 having the A317-318 mutation but retaining the WT site 2 sequence . Strikingly, immunoreactivity with this C-terminal tag is almost completely lost with Nsp3 co-expression regardless of site 1 and 2 mutations. A likely explanation for this lies in the sequence RLGGG near the C terminus of SREBP-1 .In Figure 3body-2A4 . This an1 or Leu1168 at P4 to Ala results in retention of high-molecular weight E-tag reactivity as does mutation of the P2 Gly to Ala . The loss of cutting with Ala in P2 is of note given the naturally occurring Ala in this position in site 2. As with site 2, the P5 Arg is dispensable for site 3 proteolysis . To verify our findings, we generated T7-tagged forms of SREBP-1 with C-terminal GFP tags. An ∼35 kDa Nsp3TM-specific anti-GFP reactive species is observed with either WT SREBP-1 or with mutations in sites 1 and 2. Mutation of either Gly1171 or Gly1170 to Ala results in loss of this species . No cleavage is observed at site 3 with expression of the nontransmembrane Nsp3PLpro . Similarly, Nsp3TMC111A fails to result in cleavage, while cutting is observed with Nsp3FL (A).To investigate the loss of E-tag immunoreactivity, systematic mutations to Ala of aa 1167-1171, referred to now as site 3, in the context of mutations in site 1 and site 2, were undertaken. Mutation of either Gly1171 at Ph Nsp3FL A.Figure 1 or P2 position results in loss of cutting, while neither Ser in P3 nor the Leu in P5 is required . Site 1 has a Val rather than Leu in the predicted P4 position. Mutation of this Val to Ala largely eliminates cleavage , although some residual cutting is apparent on long exposure. Together with Val being native to this position, this suggests that site 1 has a degree of flexibility beyond the canonical LXGG.We next evaluated site 1 of SREBP-1 in which site 2 and site 3 are not cleavable. As with site 3, mutation of Gly to Ala in either the predicted PA), establishing cleavage by Nsp3 after Gly 318. For site 2, T7 immunoprecipitation followed by Asp-N digestion, which cleaves before Asp, resulted in a peptide terminating at Gly 341 (DAEKLPINRLAAG) (B). These findings provide strong independent confirmation of our mutagenesis data by mass spectrometry.Establishing that there are only three sites, and determining how they can each be interrupted, allowed us to use mass spectrometry (MS) as additional proof for the noncanonical sites. Constructs were generated and mutated in the canonical site 3 and either site 1 or site 2 (single Gly to Ala mutations at 318 or 341), thus leaving only one site intact. This allowed for immunoprecipitation after cotransfection of Nsp3TM from either the N terminus of SREBP-1 through the 2 × T7 tag or the C terminus through a 3xhemagglutinin (HA) Tag, which was substituted for the E-tag. For site 1, HA immunoprecipitation followed by trypsin digestion, which cleaves peptides after Arg or Lys, results in a peptide starting at Thr 319 (TILATVPLVVDAEK) A, establINRLAAG) B. These E compare lanes 1 and 2 to lanes 5 and 6). Thus, sites 1 and site 2 are responsible for the significant Nsp3-dependent loss of SREBP-1 observed by CHX chase.To determine whether the noncanonical sites we identified are responsible for the loss of the high-molecular weight form of SREBP-1 observed by CHX chase in Fig. 4E and F and also observed in HeLa cells (A) is a consequence of Nsp3 DUB activity. However, it was recently reported that SREBP-2 has a discontinuous C-terminal degron, which is unmasked by dissociation from SCAP, allowing it to be targeted for proteasomal degradation by an unidentified ubiquitin ligase (1129IVKLGGGTAIAAS∗), would target the normally stable SREBP-2 . This truncation results in a marked stabilization of SREBP-2 (A). Mutation to KLGAA, which should prevent Nsp3-dependent cleavage of SREBP-2, did not result in SREBP-2 stabilization. These results confirm the importance of the C-terminal degron and suggest how Nsp3 could stabilize SREBP-2 by cleaving site 3 to interrupt the degron.A reasonable hypothesis is that the SREBP-2 stabilization seen on overexpression in HEK293T cells in La cells A is a con ligase . This de SREBP-2  for degr SREBP-2 . Notably SREBP-2 A, STOP.  SREBP-2 A. MutatiC111A, results in a ∼30 kDa GFP-immunoreactive band with loss of the full-length fusion protein. This is not observed with the KLGAA mutation . Notably, cytosolic Nsp3PLpro can also cleave the C terminus of SREBP-2 . The finding of SREBP-2 cleavage by soluble PLpro correlates with the SREBP-2 stabilization observed in Figure 2F. Consistent with this cleavage, SREBP-2 coimmunoprecipitates with PLpro (B), although some coimmunoprecipitation of SREBP-1 is also observed with PLpro (C), despite the lack of SREBP-1 cleavage by cytosolic PLpro.To establish whether Nsp3 interrupts the degron, we generated C-terminal GFP fusions of SREBP-2. For WT SREBP-2, coexpression of Nsp3TM, but not Nsp3TMth PLpro B, althouth PLpro C, despitAA), and the form terminating after KLGGG (STOP), by CHX chase. Nsp3TM results in a dramatic increase in the level and stability of the WT protein, similar to what is observed with the STOP mutation (D). However, even with the KLGAA mutation, where the C-terminal degron can no longer be interrupted, Nsp3TM still has a stabilizing effect . Importantly, this requires the catalytic activity of Nsp3TM. This result suggests that Nsp3 may not only be interrupting a degron by cleavage at a canonical site but also has the potential to decrease degradation of SREBP-2 by deubiquitination when the degron is mutated or inaccessible. To assess this, we examined SREBP-2 ubiquitination. When the degron is intact and the protein is normally targeted for ubiquitin-dependent degradation, significant accumulated ubiquitination is observed when proteasomal degradation is inhibited. Nsp3TM results in a loss of ubiquitination as would be expected with inactivation of the degron by PLpro cleavage . However, even with the KLGAA mutation, where the degron is no longer cleavable, a marked decrease in ubiquitination is observed with Nsp3TM , which correlates with stabilization of this mutation (D and S6D). Thus, Nsp3 can prevent SREBP-2 degradation through two mechanisms: it functions as an endoprotease to remove the degron and can potentially function as a DUB to facilitate SREBP-2 deubiquitination.To further evaluate the effect of Nsp3 on SREBP-2, we assessed WT SREBP-2 (KLGGG), the noncleavable form (KLGmutation D. Howevemutation D and S6DE), we evaluated it for cleavage when coexpressed with Nsp3TM. We observe no loss of the 145 kDa full-length species or appearance of additional fragments corresponding to N- or C-terminal halves of the protein when coexpressed with Nsp3TM (F). While we cannot exclude that SCAP may be an Nsp3 substrate in the context of SARS-CoV-2 infection, we see no evidence of cleavage under conditions where efficient cleavage of analogous sequences in SREBP-1 and SREBP-2 is observed.Our findings raise the possibility that cleavage at LXGG by the PLpro of Nsp3 might be widespread in SARS-CoV-2–infected cells, particularly in the secretory pathway. Human SCAP, but not the extensively studied hamster SCAP , containFig. S6h Nsp3TM F. While In this study, we demonstrate that SARS-CoV-2 PLpro, when expressed as a transmembrane protein, can function as a deubiquitinating enzyme for some potential ERAD substrates. Moreover, our findings establish the capacity of transmembrane forms of SARS-CoV-2 PLpro to cleave cellular ER proteins at both canonical and noncanonical sites. Most of what we observe requires PLpro to be tethered to the ER membrane. Strikingly, for canonical sites that are similarly positioned within the related SREBP-1 and SREBP-2, a requirement for membrane anchoring of PLpro was found for the former but not the latter. Our observations emphasize the importance of evaluating SARS-CoV-2 PLpro in its transmembrane ER form. They also demonstrate the need for empirical observation to identify cleavage of novel substrates, including at unpredicted sites.4 position of site 1 (LVSGG), has not been reported as being a substitute for Leu in this position. However, Ala substitutions within the canonical PLpro cleavage sites in the SARS-CoV-1 polyprotein revealed that there is a capacity for a residue other than Leu to allow for efficient cutting uniquely at the Nsp2-Nsp3 cleavage site (D). The finding that, in certain contexts, residues other than Leu in P4 still permits cutting by PLpro is consistent with structural analysis of SARS-CoV-2 PLpro bound to enzyme inhibitors, which suggests flexibility in the region that recognizes this residue (2 position (RLAAG), and where substitution with either Leu or Glu inactivates this site. Yet, substitution of Ala for Gly in the P2 position of either site 1 (LVSGG) or site 3 (RLGGG) results in the loss of detectable cutting.Cleavage of SREBP-1 at site 1 and site 2, although noncanonical and exhibiting incomplete cleavage relative to site 3, is sufficient to diminish levels of exogenously expressed SREBP-1 having an intact NTD transcription factor precursor. Val, at the Page site . In our age site D. The fi residue . A more in vitro when expressed by phage display , and differential activity of soluble PLpro toward similar sites on SREBP-1 and SREBP-2, underscore the importance of context in PLpro activity toward cellular substrates.There have been few studies examining cellular proteins that can be cleaved by PLpro. These studies have utilized the isolated PLpro and have not identified noncanonical sites or assessed site-specific requirements for cleavage. A recent study selectively assessed a region of CD177, containing a canonical PLpro consensus sequence, and found it could be cleaved by SARS-CoV-2 PLpro  display . A study display . In anot  and MycoStrip Mycoplasma Detection Kit (Invivogen) and found to be negative.HEK293T and HeLa cells, obtained from ATCC, were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C and 5% COhttps://www.addgene.org/141257/), obtained from Addgene, was a gift from Dr Fritz Roth. The full-length protein with an N-terminal 3× FLAG epitope in CMV5.1p was generated by recombination using Gateway system (Invitrogen). Plasmids encoding Nsp3 beginning at aa743, preceding the beginning of the PLpro (aa 746) and terminating either following an amphipathic helix that is immediately C terminal to the second predicted transmembrane domain at aa 1579 (Nsp3TM) or following the PLpro domain at aa 1065 (Nsp3PLpro), were generated using In Fusion (Takara) and cloned into the single N-terminal FLAG epitope–containing pCMV-Tag2B vector from BamHI to EcoRI. Mutation of the catalytic Cys to Ala  was carried out by site-specific mutagenesis .Complementary DNA encoding the full-length Nsp3 protein from the SARS-CoV-2 isolate Wuhan-Hu-1 , first cloned into the pUC57 construct from KpnI to NotI, substituting GFP for His8, and then into T7x2-SREBP-1-E from EcoRI to NotI. A construct encoding aa 1 to 520 of SREBP-1, approximating the N-terminal active transcription factor, was generated by amplifying a region corresponding to aa 410 to 520 of T7x2-SREBP-1-E and cloning this into U124AFG200-5 from HindIII to XbaI. SREBP-2 with a 3 × T7 tag at its N terminus (T7x3-SREBP-2) was synthesized by GenScript and cloned into pcDNA3.1(+)-N-HA from ClaI to NotI. GFP was inserted by subcloning a fragment beginning with an internal EcoRI site in SREBP-2 to a NotI site in the C-terminal polylinker. The eGFP was generated by PCR and first subcloned into a shuttle vector from EcoRI to XbaI. All mutations, internal deletions, and truncations in SREBP-1 and SREBP-2, except as mentioned above, were generated by site-specific mutagenesis and confirmed by sequencing.A cDNA encoding SREBP-1 isoform 1 with a 2 × T7 tag at its N terminus and an E-tag at its C terminus and silent mutations and mutations in regions critical for S1P and S2P cleavage was synthesized by GenScript and cloned into pCDNA3.1(+) from NheI to NotI (GenScript project ID: U124AFG200-5). pCDNA3.1(+) T7x2-SREBP-1-E with WT sequence, used in this study, was then generated by substituting a cDNA fragment synthesized by Integrated DNA Technologies using designed internal HindIII and XbaI sites. T7x2-SREBP-1-HA was generated by synthesis of a cDNA fragment encoding HisPlasmid encoding INSIG-1-MYC having a 6xMYC tag, HA-NHK, and concatemerized HA-Ub were gifts from Joseph Goldstein, Ron Kopito, and Dirk Bohmann, respectively , 50, 57.FLAG immunoprecipitations were with either anti-FLAG M2 Affinity Gel Beads (Sigma A222) or anti-FLAG M2 Magnetic Beads (Millipore Sigma M8823). In some experiments, normal mouse IgG (Santa Cruz Biotechnology #sc-2025) was used as a control bound to protein G MagBeads (GenScript L00274). MYC epitope immunoprecipitation was with either EZview Red anti-c-Myc Affinity Gel (Millipore Sigma E6654) or anti-FLAG M2 Magnetic Beads (Millipore Sigma M8823). FLAG immunoblots were with either monoclonal ANTI-FLAG M2-Peroxidase (HRP) produced in mouse (Sigma A8592) or goat anti-FLAG HRP (Bethyl Laboratories A190-101P). T7 immunoblots were with goat anti-T7 horseradish peroxidase (HRP) (Bethyl Laboratories A190-116P). E-tag immunoblots were with goat anti-E-tag HRP (Bethyl Laboratories A190-132P). MYC immunoblots were with 9E10  or MYC-tg for 5 to 10 min. Equal loading of protein was determined by Bradford assay. For CHX chase experiments, CHX was added to a final concentration of 50 μg/ml. In some experiments, samples were split into individual wells following transfection and β-actin served as loading control; otherwise, equal transfection efficiency was assessed using cotransfected eGFP. To assess requirements for SREBP-1 and SREBP-2 cleavage (experiments not involving CHX), cells were treated with MG132 (40 μM) for either 2 h (HEK293T) or 4 h (HeLA) prior to harvesting. For assessment of SCAP in HEK293T cells, treatment was also for 4 h. For immunoprecipitation experiments, cells were pretreated with MG132 either overnight (10 μM) or for 3 h (25 μM) prior to lysis in lysis buffer 2. After removing insoluble material by centrifugation, the clarified lysates were used for immunoprecipitation at 4 °C for 2 to 4 h, and the beads were washed three times with 10× bed volumes of lysis buffer 2. For phosphatase treatment, cells were lysed in lysis buffer 2 without EDTA or phosphatase inhibitors and incubated with 400 units of Lamba Protein Phosphatase (New England Biolabs P0753S) in a final volume of 50 μl of 1× PMP buffer and 1 mM MnCl2 (New England BioLabs kit P0753S) at 30 °C for 45 min. For assessment of INSIG-1 ubiquitination, cells were transfected with INSIG-1-MYC and HA-tagged ubiquitin. Cells were lysed in 1% SDS, heated for 1 min, and diluted with lysis buffer 1. The clarified lysate was used for immunoprecipitation, and ubiquitination was detected using anti-HA. For assessment of SREBP-2 ubiquitination, cells were transfected with T7-tagged SREBP-2 and HA-tagged ubiquitin. Cells were lysed in lysis buffer 2 for immunoprecipitation and ubiquitin detected with ubiquitin antibody.Cells were transfected with the indicated plasmids for 30 to 40 h using either PolyFect (Qiagen) or JetPrime (Polyplus 1010000046). For assessment of protein levels and protein loss, cells were lysed either immediately or at the indicated times in lysis buffer 1: PBS pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate (INSIG-1 and NHK) or in lysis buffer 2: 10 mM Tris–HCL pH 8, 140 mM NaCl, 1% Triton X-100, 0.1% SDS , supplemented with 1 mM EDTA and phosphatase inhibitor . Lysis buffers were supplemented with 20 to 40 μM MG132 (APE × BIO A2528), 10 mM iodoacetamide, and cOmplete Protease Inhibitor Cocktail EDTA-free (Roche 11836153001). Insoluble material was removed by centrifugation at >15,000×Immunoprecipitates and clarified cell lysates were denatured in reducing lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific NP008), followed by resolution by SDS-PAGE and transfer to nitrocellulose membranes (Thermo Fisher Scientific 88018). Immunoblots were developed either using primary antibodies followed by HRP-conjugated secondary antibodies or primary antibodies directly conjugated to HRP followed by enhanced chemiluminescence using SuperSignal West Pico Plus (Thermo Fisher Scientific 1863096) or Clarity Western ECL (Bio-Rad 170-5061). Images were obtained either on films or by using an Azure Biosystems c280 imager.HEK293T cells, grown in 10 cm dishes, were cotransfected with plasmid encoding Nsp3TM and T7x2-SREBP-1-HA having a mutated site 3 (KLGGA) and either a mutated site 1 (LVSGA) or site 2 (RLAAA). After 24 h, samples were treated with 40 μM MG132 for 2 h, lysed in lysis buffer 2, and soluble material immunoprecipitated sequentially with T7 agarose beads (EMD Millipore), followed by Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific).After extensive washing, beads were resuspended in 25 mM Hepes, pH 8 and heated at 95 °C for 5 min to denature proteins. The samples were digested overnight with either 2 μg of trypsin (Promega) or 2 μg of Asp-N (Promega) at 37 °C. The supernatants containing protease-digested peptides were collected after centrifugation of beads and the peptides cleaned with the EasyPep micro sample preparation kit  and lyophilized.6 over a mass range of 400 to 1600 m/z, followed by tandem mass spectrometry analysis at a resolution of 15,000 with an ion accumulation target set at 5e5. Fragment precursor isolation width was setup at 1.6 m/z, normalized collision energy was 30, and charge state 1 and unassigned charge states were excluded. Acquired MS/MS spectra were searched against human UniProt protein database using a SEQUEST and percolator validator algorithms in the Proteome Discoverer 2.4 software (Thermo Fisher Scientific) . Enzyme was specified as Trypsin (set as Semi tryptic) or Asp-N. The precursor ion tolerance was set at 10 ppm, and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation as dynamic modification. A false discovery of 0.01 and a minimum peptide length of six amino acids were used for peptide identification.The peptides were resuspended in 0.1% TFA and subjected to nanoflow liquid chromatography  coupled to high-resolution tandem MS . MS scans were performed in the Orbitrap analyzer at a resolution of 120,000 with an ion accumulation target set at 4eCells were plated at a confluence of ∼60% into 12-well plates containing 18 mm diameter cover slips (Ted Pella 26022) that had been pretreated with 50 mM poly-D-lysine (Sigma P1024). After 5 h, cells were transfected with indicated plasmids using JetPrime. After 24 h, cells were fixed for 10 min in 4% paraformaldehyde at room temperature and permeabilized for 5 min in 100% methanol precooled to −30 °C. The coverslips were then washed with PBS pH 7.4 containing 0.05% Tween 20 (PBST) and treated with buffer F (PBST supplemented with 5% goat serum) for 1 h to reduce nonspecific absorption. After washing three times with PBST, coverslips were incubated with the indicated primary antibodies in buffer F for 1 h at room temperature, washed three times with PBST, and then incubated with secondary antibodies for 1 h at room temperature. After washing three times with PBST, coverslips were mounted onto a glass slide in mounting medium  for imaging.Widefield images were acquired using inverted Eclipse Ti2 inverted microscope (Nikon Inc), equipped with or an ORCA-Flash4.0 V3 (Hamamatsu), Lumencor SPECTRA light engine (Lumencor Inc), 100× NA 1.42 and 1.45 Plan Apo objective, and 1.5× magnifying tube lens. 200-nm thick Z sections were acquired.https://fiji.sc/) and Photoshop (Adobe) were used to assemble image panels. AutoQuant X3 software (MediaCybernetics) (https://www.meyerinst.com/mediacybernetics/autoquant/) was used for deconvolution. Image panels show one representative Z plane.Structured illumination microscopy (SIM) was performed on N-SIM (Nikon Inc), equipped with 405, 488, 561, and 640 nm excitation lasers, Apo TIRF 100× NA 1.49 Plan Apo oil objective, and back-illuminated 16 μm pixel EMCCD camera . 100-nm thick Z Sections were acquired in 3D SIM mode and reconstructed. Fiji  (https://www.graphpad.com/) to generate plot profiles.To illustrate the typical pattern of signal overlap from microscopy images, line plots were drawn across multicolor images as indicated in the figure panels. The plot profile function in Fiji (ImageJ2) was used to determine the intensities of the fluorescent signals along the drawn line. The data was exported to Prism (GraphPad) (MS data is available on the MassIVE server reference number: MSV000092963. All other data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.