The FEBS Journal

Bacterial deubiquitinases (DUBs) are important virulence effectors that manipulate host ubiquitin signaling during infection. ElaD, a CE-clan DUB expressed by enterohemorrhagic Escherichia coli, preferentially cleaves K63-linked ubiquitin chains, yet its effects on conserved cellular stress responses remain poorly understood. We demonstrate that ElaD exhibits redox-dependent DUB activity in vitro. In addition, we identified the molecular basis underlying the selective recognition of substrate proteins, ubiquitin and NEDD8 by ElaD. Structural and mutational analyses reveal that, beyond the conserved catalytic site, ElaD engages ubiquitin through a combination of electrostatic and hydrophobic interactions. Using Saccharomyces cerevisiae as a heterologous model system, we show that wild-type ElaD rescues the proteotoxic stress phenotype of ubp2{Delta} yeast cells, whereas specific ElaD mutants fail to confer a similar response. Furthermore, expression of ElaD suppresses oxidative stress-induced accumulation of K63-linked polyubiquitin and may perturb stress-associated translational regulation linked to K63 ubiquitin signaling. Consequently, cells expressing ElaD exhibit altered stress adaptation and diminished fitness during prolonged oxidative stress. Collectively, these findings indicate that ElaD perturbs ubiquitin-mediated stress signaling by counteracting K63-linked ubiquitination events that support adaptive cellular responses. Our study highlights how a bacterial DUB can reprogram conserved ubiquitin-dependent pathways and exploit host ubiquitin signaling networks to modulate cellular stress responses and protein homeostasis. These findings further suggest potential host targets of bacterial DUBs during infection.

Insulin-degrading enzyme (IDE, insulysin, insulinase) is a peptidase that hydrolyzes a number of bioactive peptides including insulin and the amyloid beta peptide, making it a promising therapeutic target for diabetes and Alzheimers disease. Aspects of its physiological role are still controversial, however. In an attempt to further define IDEs role in cells, we used co-immunoprecipitation experiments to identify potential IDE interacting proteins. The enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) was found associated with IDE in three different cell lines, and the two proteins colocalize in HeLa cells. Purified PYCR1 activates IDE toward small peptide substrates, suggesting a modulatory role for the interaction in vivo. Modeling suggests that the unstructured N-terminal region of PYCR1 inserts into allosteric sites of IDE, contributing to the observed activation. Deleting this sequence alters, but does eliminate, the interaction between PYCR1 and IDE. Since pyrroline-5-carboxylate reductase 1 is a mitochondrial protein, we posit that their interaction could regulate a previously described mitochondrial pool of IDE, which may serve to degrade mitochondrial targeting sequences or amyloid beta peptide that localizes to that organelle.

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.

Fic enzymes mediate diverse post-translational modifications, including adenosine monophosphate (AMP) transfer and removal, referred to as AMPylation and deAMPylation, respectively. We identified the prokaryotic translation elongation factor Tu (EF-Tu) as an AMPylation target of the Fic enzyme SoFic. SoFic can constitutively reverse EF-Tu modification via deAMPylation whereas AMPylation depends on SoFic homodimerization. The complex crystal structure between SoFic and EF-Tu confirms a conserved target binding mode across evolutionary distant Fic enzymes. AMPylation disrupts EF-Tu's regulatory switch-I region, causing translational inhibition. SoFic furthermore binds to its promotor DNA, suggesting a dual function as transcriptional and translational regulator in bacterial cells. Together, our structural and biochemical data provide valuable insights into the functional and regulatory diversity of Fic enzymes.

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.

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.

KANK proteins link integrin adhesions to the cortical microtubule stabilising complex (CMSC) through interactions with the adhesion adaptor talin. However, how KANK proteins are regulated remains unclear. Here we show that the KN domain of KANK proteins contains separable regions that mediate talin binding and a conserved intramolecular interaction. Using fluorescence polarisation, NMR spectroscopy and structural analysis, we map an interaction between the N-terminal KN domain and the C-terminal ankyrin repeat domain and identify residues 60-68 of the KN domain as required for this intramolecular interaction. In contrast, the canonical LD motif within residues 30-60 mediates binding to talin. Deletion of residues 60-68 disrupts the intramolecular interaction while preserving talin binding, demonstrating that the KN domain contains distinct modules for talin engagement and intramolecular regulation. This regulatory architecture is conserved across the KANK family, although sequence variation modulates the strength of the intramolecular interaction. Together, these findings identify a modular organisation within the KANK KN domain that separates talin recognition from intramolecular regulation and is consistent with an autoinhibitory mechanism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/731086v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@142543eorg.highwire.dtl.DTLVardef@1a8e454org.highwire.dtl.DTLVardef@1266c93org.highwire.dtl.DTLVardef@1a25fe7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules.The LD motif (blue) mediates binding to the talin R7 domain, whereas residues 60-68 (yellow) are required for interaction with the C-terminal ankyrin repeat domain. An AlphaFold model is shown as a structural interpretation of the intramolecular KN-ankyrin repeat interaction identified in this study. C_FIG

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.

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.

Membrane-bound decarboxylases couple carboxylic acid decarboxylation to the transport of Na+ ions out of prokaryotic cells. The molecular mechanism of decarboxylase action is not yet known, which contrasts with the progress achieved in studying other primary ion pumps. Measuring decarboxylase activity is complicated by slow keto-enol tautomerization of the substrates during the assay. We found that HEPES exhibits anomalously high efficiency as a general acid catalyst for C-H bond formation during the enol-to-ketone conversion of oxaloacetate. Accordingly, the addition of HEPES to the assay medium eliminated the contribution of tautomerization rate to measured decarboxylation rate. Using the dependence of oxaloacetate tautomerization rate and equilibrium on solvent properties and pH, we established that only the keto form of oxaloacetate is converted by Vibrio cholerae oxaloacetate decarboxylase. Steady-state kinetic measurements did not reveal cooperativity in oxaloacetate conversion and Na+ binding. The effects of ionophores (CCCP, valinomycin, and ETH157) on proton transport in pyranine-loaded membrane vesicles prepared from V. cholerae cells indicated that the proton required for the conversion of oxaloacetate to pyruvate is taken up from the cytoplasmic side of the membrane. Furthermore, the effects suggested that {Delta}pH generation is caused by secondary electrophoretic proton transport in exchange for Na+.These findings advance our understanding of the molecular mechanism of the decarboxylation-supported Na+ transport in bacteria.

Angiotensin-converting enzyme 2 (ACE2) functions as the receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the cellular endocytic machinery for entry by binding to defined residues on ACE2 with its spike protein (S protein), whose activation requires a priming process by another transmembrane protease, the transmembrane protease serine 2 (TMPRSS2). In addition, ACE2 itself is cleaved by TMPRSS2, which has been shown to be critical for viral pathology. This study aimed to elucidate the relationship between ACE2 and TMPRSS2 and the mechanism of ACE2 processing under normal cellular conditions. It is shown that interaction of ACE2 with TMPRSS2 results in altered processing, modification and cellular localization. Glycosylation of ACE2 has a major impact on TMPRSS2 interaction, trafficking and shedding of the enzyme. Studies in newly generated TMPRSS2-knockout rats reveal increased ACE2 levels in tissues supporting an important role of TMPRSS2 in ACE2 shedding also in vivo.

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.

Androglobin (ADGB), a protein essential for spermatogenesis, is the most structurally unusual member of the vertebrate globin superfamily. It combines a calpain-like domain with a circularly permuted globin domain containing an embedded calmodulin-binding IQ motif, an architecture suggesting complex regulatory functions that remain poorly understood. Here, we investigated whether ADGB undergoes calcium-dependent post-translational processing, like it has been described for other calpains. ADGB underwent robust proteolytic processing upon calcium stimulation, generating several stable cleavage products following ectopic expression in mammalian cells. In vitro proteolysis assays demonstrated that ADGB cleavage requires cytoplasmic factor(s) and is strongly enhanced by Ca2+. While this process is sensitive to pan-calpain inhibition, siRNA-mediated knockdown excluded calpain 1 (CAPN1) and calpain 2 (CAPN2) as primary mediators of ADGB cleavage. In contrast, depletion of the calpain small regulatory subunit CAPNS1 markedly reduced calcium-dependent ADGB proteolysis, implicating a CAPNS1-associated calcium-responsive proteolytic pathway. Domain-mapping analyses localized the major cleavage hotspot between the N-terminal calpain-like domain and the globin-containing C-terminal region, indicating that proteolysis separates the protease-like and globin modules of the ADGB chimera. The isolated globin domain displayed enhanced interaction with calmodulin compared with full-length ADGB, whereas the extended C-terminal region impeded this interaction. Furthermore, unlike full-length ADGB, the isolated globin domain exhibited preferential localization to centrosomal structures. Collectively, these findings identify calcium-dependent proteolysis and altered subcellular localization of the isolated globin domain as previously unrecognized properties of ADGB that may be relevant to its role in ciliary biology.

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.

Pulmonary fibrosis is characterized by extracellular matrix (ECM) deposition driven by fibroblast-to-myofibroblast transition (FMT) and by an altered proteolytic balance. While the roles of several cysteine proteases have been documented, the specific contribution of cathepsin V (CatV) and legumain (LGMN) remains poorly explored. LGMN, CatV and their dual inhibitor cystatin M/E (CysM/E) are significantly increased in lung specimens and bronchoalveolar lavage fluids from patients with idiopathic pulmonary fibrosis. TGF-{beta}1 triggered CysM/E expression and LGMN transcription, intracellular maturation, enzymatic activity, and pro-LGMN secretion via the Smad-3 pathway, whereas CatV was downregulated in human lung fibroblasts (CCD-19Lu and primary HPF cells) undergoing myodifferentiation. Genetic silencing of LGMN or CatV, and pharmacological inhibition of LGMN, led to accumulation of fibronectin and elastin, implying that both proteases contribute to ECM remodeling. LGMN cleaved fibronectin, while CatV predominantly regulated elastin levels. Conversely, broad-spectrum inhibitor cystatin C (hCC) markedly reduced elastin and fibronectin degradation, whereas CysM/E exerted a weaker effect, mainly on elastin turnover. LGMN inhibition transiently delayed fibroblast wound closure, establishing a functional role in tissue repair through fibronectin remodeling. Neither LGMN nor CatV influenced -SMA expression, distinguishing them from CatB, which participates in FMT. Altogether, LGMN was identified as an effector of matrix remodeling rather than myodifferentiation, acting in concert with CatV. Within the proteolytic network governing fibrosis progression, the present findings identify a cystatin-regulated LGMN/CatV partnership, participating in ECM turnover and cell migration. Present results also provide new perspectives on potential therapeutic protease-based strategies targeting ECM turnover underlying lung fibrosis.