Structural basis for effector recognition by an antibacterial type IV secretion system

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD–VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD. Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

BMP1 is Not Required for Lung Fibrosis in Mice

Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as “methylation”). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as methylation). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

A Two-Year Clinical Description of a Patient with a Rare Type of Low-GGT Cholestasis Caused by a Novel Variant of USP53

Here, we report a novel truncating mutation in the ubiquitin-specific peptidase gene (USP53) causing low-γ-GT (GGT) cholestasis. Genetic testing was carried out, including clinical exome sequencing for the proband and Sanger sequencing for the proband and his parents. The proband harbored a novel c.1017_1057del (p.(Cys339TrpfsTer7)) mutation in the ubiquitin carboxyl-terminal hydrolase (UCH) domain of USP53; we describe the clinical and laboratory features of the patient with a rare type of low-GGT cholestasis caused by this variant. The clinical presentation was found to be similar to that of phenotypes described in previous studies. However, there was an unusual presence of liver hemangiomas observed in our patient. Thus, our report reinforces the link between USP53 mutations and cholestasis. With this report, we confirm USP53 as the gene for low-GGT cholestasis and describe liver hemangiomas as a possible additional symptom of the phenotype spectrum. The inclusion of USP53 in the OMIM database and liver gene panels can further increase the effectiveness of molecular genetic studies.

Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that assisting CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa . CtpA copurifies with the uncharacterized lipoprotein, LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis, and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the LbcA copurification partners are not cleaved by CtpA, but might copurify with LbcA because they participate in multi-enzyme complexes containing CtpA substrates. These findings are important, because CTPs and their associated proteins affect peptidoglycan remodeling and virulence in multiple species.

MICAL1 activation by PAK1 mediates actin filament disassembly

SummaryThe MICAL1 monooxygenase has emerged as an important regulator of filamentous actin (F-actin) structures that contribute to numerous processes including nervous system development, cell morphology, motility, viability and cytokinesis [1–4]. Activating MICAL1 mutations have been linked with autosomal-dominant lateral temporal epilepsy, a genetic syndrome characterized by focal seizures with auditory symptoms [5], emphasizing the need for tight control of MICAL1 activity. F-actin binding to MICAL1 stimulates catalytic activity, resulting in the oxidation of actin methionine residues that promote F-actin disassembly [6, 7]. Although MICAL1 has been shown to be regulated via interactions of the autoinhibitory carboxyl-terminal coiled-coil region [8] with RAB8, RAB10 and RAB35 GTPases [9–12], or Plexin transmembrane receptors [13, 14], a mechanistic link between the RHO GTPase signaling pathways that control actin cytoskeleton dynamics and the regulation of MICAL1 activity had not been established. Here we show that the CDC42 GTPase effector PAK1 serine/threonine kinase associates with and phosphorylates MICAL1 on serine 817 (Ser817) and 960 (Ser960) residues, leading to accelerated F-actin disassembly. Deletion analysis mapped PAK1 binding to the amino-terminal catalytic monooxygenase and calponin domains, distinct from the carboxyl-terminal proteinprotein interaction domain. Stimulation of cells with extracellular ligands including basic fibroblast growth factor (FGF2) led to significant PAK-dependent Ser960 phosphorylation, thus linking extracellular signals to MICAL1 phosphorylation. Moreover, mass spectrometry analysis revealed that co-expression of MICAL1 with CDC42 and active PAK1 resulted in hundreds of proteins increasing their association with MICAL1, including the previously described MICAL1-interacting protein RAB10 [15]. These results provide the first insight into a redox-mediated actin disassembly pathway linking extracellular signals to cytoskeleton regulation via a RHO GTPase family member, and reveal a novel means of communication between RHO and RAB GTPase signaling pathways.