The FEBS Journal

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron-sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min-1--a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186-199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

Enzyme activity increases with temperature up to a maximum, beyond which it declines, a behaviour traditionally attributed to thermal denaturation. However, some enzymes show activity decline well below the melting temperature. Macromolecular rate theory (MMRT) explains this phenomenon by introducing a negative activation heat capacity [Formula], reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, [Formula] has been shown to be temperature-dependent and proposed as a general catalytic feature, though its variation within and across homologous families from distinct thermal niches remains unexplored. We characterized the glucokinase activity of three homologous bifunctional ADP-dependent PFK/GK enzymes: MbPFK/GK from the psychrotolerant Methanococcoides burtonii, MmPFK/GK from the mesophilic Methanococcus maripaludis, and ancM, the inferred ancestor of the Methanococcales order, which displays enhanced thermostability. MmPFK/GK and ancM display two [Formula] regimes, with abrupt changes in kcat vs temperature: zero to moderately negative values at low temperatures, shifting sharply at elevated temperatures to highly negative values (-44 kJ mol-1 K-1 and -36 kJ mol-1 K-1, respectively), exceeding previous reports. Circular dichroism spectroscopy confirms that these extreme values reflect pre-melting conformational changes rather than denaturation. Despite being psychrotolerant, MbPFK/GK displayed the highest thermal stability [Formula] and a single [Formula] regime throughout all temperatures (-2.6 kJ mol-1 K-1). Domain-closure dynamics explain thermal adaptation and moderate-temperature [Formula] values; whereas the basis of the extreme high-temperature [Formula] values remain unknown. To account for these two regimes, we present a two-pathway model incorporating a conformational equilibrium in which free enzyme and enzyme-substrate complex populate two catalytically competent conformations.

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.

Anaerobic ammonium-oxidizing (anammox) bacteria are essential players in the global nitrogen cycle, responsible for converting ammonium and nitrite directly to nitrogen gas. Anammox bacteria have unique features such as a specialized cellular compartment - the anammoxosome. Candidatus (Ca.) Brocadia pituitae genome, as other anammox bacteria, encodes for a diversity of hydroxylamine oxidoreductase (HAO) paralogs, often pointed out as the enzymes responsible for most of the reactions of the anammox cycle. One of this Ca. B. pituitae HAO paralogs is an 840-amino acids protein, named here as BpMHAO, that stands out for its unprecedented domain organization, which includes a multicopper oxidase-like (MCo-like) domain followed by the HAO-like one. Sequence and structural analyses classified this MCo-like domain as homologous to the small laccase family. Spectroscopic characterization revealed a distinct UV-visible spectrum, tentatively assigned to the T3 center, whereas the EPR spectra confirmed the presence of T1, T2 and T3 copper centers. Enzymatic studies demonstrated limited laccase and oxygen-dependent ferroxidase activities. On the other hand, enzymatic assays performed in cell extracts from Escherichia coli and Shewanella oneidensis, harbouring the recombinant HAO-like domain, exhibited a robust hydroxylamine reductase activity using methyl viologen as the electron donor. Our results showed that the BpMHAO potentially plays a role in the anammox process/reactions by converting hydroxylamine into hydrazine. This feature can be relevant to anammox bacteria either by i) mitigating unwanted hydroxylamine, obtained by incorrect formation of this compound, by converting it into hydrazine and enabling its use in the anammox reaction or ii) using hydroxylamine from the outside medium as a substitute for ammonium, delivering hydrazine directly to the last step of the cycle, short-circuiting its first steps.

SUMOylation is an essential eukaryotic ubiquitin-like post-translational modification that plays a central role in the regulation of various nuclear processes and stress responses. It canonically occurs at lysine residues within {Psi}KXE consensus motifs that lie in intrinsically disordered regions or loops and interact specifically with the SUMO-conjugating E2 enzyme UBC9. However, many detected SUMOylation sites are found within structured domains, and it remains unclear how these are recognised by UBC9. Here, we investigated the SUMOylation of Lys43 in the BTB domain of human ZBTB38 (ZBTB38BTB), a lysine located within a rigid {beta}-sheet. By combining X-ray crystallography, structural prediction, and in-vitro UBC9 interaction and SUMOylation assays, we show that ZBTB38BTB possesses a dedicated surface that recapitulates the spatial arrangement of residues found in canonical linear consensus motifs. This surface binds UBC9 with mid-micromolar affinity and is predicted to position Lys43 in its active site for efficient SUMOylation. Structural modelling and sequence analyses suggest that this property is shared by BTB domains of five members (10%) of the ZBTB-protein family across vertebrates, revealing a previously unrecognised property of a subset of ZBTBBTB domains. Kinetic analyses reveal that, under the reaction conditions used, the catalytic efficiency of ZBTB38BTB and ZBTB33BTB SUMOylation are closely comparable to that of the C-terminal domain of RANGAP1, the best-characterised and most efficiently SUMOylated substrate known. This defines a new class of inherently efficient, E3 ligase-independent SUMOylation substrates beyond RANGAP1 and suggests that structural pre-organisation of the acceptor lysine and its environment may promote productive UBC9 engagement. Lastly, we demonstrate the presence of higher-molecular-weight, modified forms of ZBTB38 in human cells, consistent with SUMOylation. Together, these results provide a biochemical basis for interpreting existing and designing future studies on the functional impact of ZBTB SUMOylation. More broadly, our findings offer insights into the determinants of efficient SUMOylation, and may facilitate the identification of further inherently efficient targets, and, potentially, the design of SUMOylation modulators.

The synthesis of mitochondrial-encoded polypeptides is an essential process, primarily regulated at the posttranscriptional level. In yeast, many regulatory factors have been described as acting in proximity to the mitoribosome to promote efficient translation; however, the precise mechanisms by which these components function remain largely unknown. Here, we expand on findings concerning a previously studied mitoribosome interactor, Mrx9, which is found in large expressosome-like assemblies of mitoribosome clusters. Mrx9 was initially linked to mitochondrial translation and was suggested to be associated with the splicing of COX1 and COB transcripts. Our current data show that Mrx9 is associated with the PHB/m-AAA complex at the polypeptide exit tunnel of the mitoribosome. Overexpression of Mrx9 impairs the proteolytic functions of Yta10 and Yta12 within the prohibitin complex, leading to splicing defects; accumulation of aberrant polypeptides; and a noticeable impairment in the processing of the essential mitoribosomal protein bL32m. These findings support a regulatory role for Mrx9 in the PHB/m-AAA complex by modulating the activities of both Yta10 and Yta12.

The fungal bioluminescence pathway enables autonomous light production from caffeic acid and has emerged as a versatile platform for bioimaging and synthetic biology. However, biochemical characterisation of its core enzymes remains limited. Here, we establish recombinant purification and quantitative in vitro characterisation of fungal luciferase (Luz) and hispidin-3-hydroxylase (H3H) homologues from Neonothopanus nambi and Mycena chlorophos. Complementary luminescence and mass spectrometry-based assays revealed pronounced substrate inhibition of H3H by hispidin, conserved across both homologues. Direct monitoring of substrate turnover further revealed an unexpected secondary H3H-catalysed conversion of 3-hydroxyhispidin that remained dependent on NADPH and FAD. NMR, isotope labelling and product characterisation support an additional oxidative transformation followed by formation of multiple downstream products, including caffeic acid, thereby reconnecting secondary turnover with an upstream intermediate of the pathway. H3H nevertheless retained a strong kinetic preference for hispidin over 3-hydroxyhispidin. Together, these findings reveal previously unrecognised catalytic complexity within the fungal bioluminescence pathway and provide a biochemical framework for understanding and engineering pathway flux.

Macroautophagy/autophagy is a process that degrades intracellular components and is strongly triggered by nitrogen starvation (-N). Some ATG (autophagy related) genes are activated at the transcriptional level in nitrogen starvation; however, a full understanding of transcriptional induction and the role of chromatin during this process remains unclear. To address this, we measured the occupancy of RNA polymerase II (Pol II), histone H3, and acetylated H4 (H4Ac) under nutrient-rich and -N conditions by ChIP-seq. We found that most genes are rapidly downregulated within 15-30 min, including ribosomal protein (RP) and biogenesis (RiBi) genes. Meanwhile, genes involved in amino acid (AA) biosynthesis are upregulated, along with many ATG genes. Unexpectedly, RP and RiBi genes were reinduced by 3 hours. Furthermore, many upregulated genes remained active during prolonged starvation. Histones are typically removed from promoters during transcription activation. Consistent with this, we found that most induced genes exhibited histone eviction and increased H4 acetylation at their promoters, suggesting a possible role for histone acetylation in their activation. In line with this, depleting Esa1, an essential H4 histone acetyltransferase, nearly abolished the induction of ribosomal biosynthetic genes and many AA biosynthetic genes. Sustained activation of many genes during prolonged starvation highlights the vital role of transcription in supporting autophagy and cell survival. This is the first comprehensive study to detail changes in chromatin, histone acetylation, and transcription during nitrogen starvation, highlighting the importance of Esa1 and H4Ac in this process.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

Lipoate is an essential protein-bound cofactor attached to conserved lysine residues in structurally related acceptor proteins, including GcvH of the glycine cleavage system and LbpA proteins required for sulfur oxidation by the sulfur-oxidizing heterodisulfide-like (sHdr) complex. The coexistence of distinct lipoylation pathways and acceptor proteins raises the question of how the enzymes initiating lipoate attachment discriminate between homologous but functionally distinct substrates. We systematically tested lipoate:protein ligases and potential acceptor proteins from five representatives of the bacterial phyla Pseudomonadota and Aquificota using native gel-shift assays and MALDI-TOF mass spectrometry. The canonical Escherichia coli lipoate:protein ligase Lpl(AB) preferentially modified GcvH proteins but showed little or no activity toward LbpAs. In contrast, sulfur oxidation-associated lipoate:protein ligases, termed sLpl(AB), which function in the sLpl(AB)-LipS1/LipS2 lipoate assembly pathway, strongly preferred LbpA proteins and generally failed to modify GcvH. Conserved cysteine residues characteristic of LbpAs were dispensable for recognition by sLpl(AB). Instead, comparison of amino acid sequences and electrostatic surfaces revealed predominantly acidic interaction surfaces in GcvH proteins and corresponding basic regions in LbpAs. Accordingly, replacement of selected basic regions in Roseovarius mucosus LbpA2 by acidic residues reduced modification by its cognate sLpl(AB) and simultaneously enabled weak modification by E. coli Lpl(AB). These findings establish electrostatic surface properties as major determinants of substrate discrimination by lipoate ligases and show that recognition depends on distributed features of the folded acceptor protein rather than solely on the sequence surrounding the lipoylated lysine.

NF-{kappa}B essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKK and IKK{beta} to NF-{kappa}B signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKK KO cells than IKK{beta} KO, whilst IKK+IKK{beta} double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-{kappa}B activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKK or IKK{beta}. Finally, re-expression of IKK increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-{kappa}B signalling by disrupting NEMO:IKK interactions.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide, yet therapeutic options remain limited. TMPRSS6, a liver serine protease best known for its role in iron homeostasis, has recently emerged as a potential therapeutic target for MASLD. However, the molecular mechanisms linking TMPRSS6 to hepatic lipid metabolism remain incompletely understood. To identify novel TMPRSS6 substrates, we performed extracellular proteomic analyses of TMPRSS6-overexpressing cells. Among the proteins identified, {beta}-klotho (KLB), a co-receptor required for FGF19 and FGF21 signaling, emerged as a compelling candidate substrate. We demonstrate that TMPRSS6 interacts with KLB and promotes its proteolytic shedding in a catalytic activity-dependent manner. Functionally, TMPRSS6 reduced full-length KLB abundance at the cell surface and attenuated FGF19-dependent FGFR4 signaling in a heterologous expression system. Together, these findings identify KLB as a novel functional substrate of TMPRSS6, providing a mechanistic framework through which this protease may influence hepatic lipid metabolism. These results provide a rationale for investigating the regulation of KLB and other candidate substrates by TMPRSS6 in physiological models and further support its evaluation as a therapeutic target for MASLD.

Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of an endoplasmic reticulum-specific pentose phosphate pathway, regenerating luminal NADPH levels in the process. Its function remains insufficiently well understood. Since expression of H6PD is notably high in the liver, we aimed to assess its role in hepatic metabolism. Considering the central role of the liver in lipid synthesis, breakdown and storage, we focused our efforts specifically on studying the effect of H6PD on hepatic lipid metabolism. An H6PD knockout mice strain was generated and characterized by liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based lipidomic and proteomic analyses of liver tissue. Lipidomics analysis revealed an overall increase in hepatic triglycerides and a specific increase in unsaturated long-chain triglycerides in H6PD knockout mice. Intracellular lipid accumulation was confirmed through Nile Red staining of liver sections. Functional enrichment analysis of proteomics data from the H6PD deficient mice identified a corresponding upregulation of multiple fatty acid metabolism-associated pathways. Additionally, an H6PD knockout AML12 cell line was generated through CRISPR/Cas9 and characterized by lipid staining and functional assays to assess metabolic outcomes. Loss of H6PD led to intracellular lipid accumulation, reduced mitochondrial {beta}-oxidation and increased sensitivity to lipotoxicity, even though fatty acids remained the cells' primary mitochondrial fuel. Ultimately, our results indicate that H6PD plays an as-of-yet undescribed role in hepatic lipid metabolism, implying a link between the availability of NADPH within the endoplasmic reticulum and fatty acid homeostasis.

Phosphatidic acid (PA) phosphatase (PAP) is an enzyme that plays a major role in lipid signaling by controlling the cellular levels of two lipid secondary messengers: its substrate, PA, and its product, diacylglycerol. Two types of mammalian PAPs have been reported to date. Type 1 PAP (PAP1) is an Mg2+-dependent, N-ethylmaleimide (NEM)-sensitive cytosolic enzyme (EC 3.1.3.4), whereas type 2 PAP (PAP2), also known as phospholipid phosphate (PLPP) (EC 3.1.3.113), is an Mg2+-independent, NEM-insensitive transmembrane protein. PAP2 also hydrolyzes other bioactive lipids such as lyso-PA (LPA), sphingosine-1-phosphate (S1P), and ceramide-1-phosphate (C1P). Here, we purified human phosphatase orphan 2 (PHOSPHO2), a putative cytosolic phosphatase containing a haloacid dehalogenase-like domain, and characterized its enzymological properties in vitro. Purified PHOSPHO2 displays Mg2+-dependent, NEM-sensitive phosphatase activities toward PA, LPA, S1P, C1P, and glycerol-3-phosphate (G3P) in vitro. Moreover, PHOSPHO2 showed substrate selectivity for PA molecular species containing shorter saturated fatty acids such as lauric acid and myristic acid, or polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid. The PAP activity of PHOSPHO2, but not its other phosphatase activities, was strongly enhanced in the presence of phosphatidylcholine and phosphatidylethanolamine, major components of the cell membranes. These results indicate that mammalian PHOSPHO2 is a novel cytosolic PLPP that primarily functions as a PAP on cytoplasm-facing membranes.

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

Background. Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods. Male, 8 weeks-old mice bearing phosphatase and tensin homolog (Pten) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results. Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid {beta}-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and -linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions. Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation.

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.