Biochemical Journal最新文献

Bacterial caseinolytic protease (Clp) chaperone-protease complexes are essential for the degradation of misfolded and aggregated protein substrates. The spirochaete Leptospira interrogans possesses a set of Clp adaptor proteins (ClpS1 and ClpS2) and chaperones (ClpX, ClpA and ClpC), which are believed to associate with two distinct isoforms of ClpP (ClpP1 and ClpP2). This study explores the structural and functional properties of LinClpA, LinClpS1 and LinClpS2 derived from L. interrogans. LinClpA, a 740-amino acid protein, features an N-terminal domain and two AAA+ ATPase domains (D-I and D-II), containing conserved motifs critical for ATP binding and hydrolysis. LinClpS1 and LinClpS2 exhibit similar structures, yet they possess distinct binding pockets for N-degron substrates. Biochemical assays indicate that the N-domain-deleted variant of LinClpA (LinClpAΔN) exhibits a nucleotide-induced oligomerization tendency similar to LinClpA's but demonstrates higher ATPase activity. Interaction studies have shown that LinClpA's ATPase activity is enhanced in the presence of LinClpP isoforms and inhibited by LinClpS isoforms. In contrast, the activity of LinClpAΔN remained unaffected by LinClpS1 and LinClpS2, highlighting the significance of the N-domain of LinClpA in adaptor protein interactions. Furthermore, the study predicted and evaluated the role of the C-degron tag called small stable RNA A in facilitating protein degradation by the L. interrogans ClpAP1P2 machinery.

Earlier, we showed that jumonji domain containing protein 6 (JMJD6) interacted with HOTAIR promoter (-123 to -103 bp, termed JMJD6 interaction region [JIR]) and for maximal induction, an additional (-216 to -123 bp) region was required. In silico prediction and ENCODE data from MCF7 cells showed Y-box interacting protein 1 (YBX1) peaks in this region (YIR). Publicly available mass spectrometry data of proteins following JMJD6 immunoprecipitation identified YBX1 as an interacting partner. In this study, we validate JMJD6-YBX1 interaction in breast cancer cell lines using co-immunoprecipitation assays with recombinant, endogenous and in vitro synthesized proteins. Domain mapping using deletion constructs revealed that the A/P domain of YBX1 interacted with the JMJC domain of JMJD6. These proteins also positively regulated each other's expression in breast cancer cell lines. Further, YBX1 augmented luciferase activity of HOTAIR promoter constructs, pHP216 and pHP123, in MCF7, Vec and JMJD6 overexpressing cells. siRNA-mediated depletion, mutation of YIR region or knocking out YBX1 (YKO cells) diminished luciferase activity. ChIP and ChIP-re-ChIP assays verified co-occupancy of both proteins in the HOTAIR promoter region. Electrophoretic mobility shift assays confirmed complex formation with YIR and JIR probes. Mutation of the YIR region and YKO resulted in loss of complex formation with both probes. Taken together, these data imply that YBX1 is crucial for physically recruiting JMJD6 to the HOTAIR promoter. Their interaction and positive feed-forward loop, perpetuated by JMJD6 and YBX1 inter-regulation, culminates in HOTAIR induction, which in turn is known to drive tumour progression.

The Rab GTPase switch-2 region is a hotspot for post-translational modifications. Its phosphorylation can determine whether individuals develop Parkinson's disease or not. Other modifications of the same region are catalyzed by enzymes from bacterial pathogens when they infect human cells. Here, we profiled a set of kinases including LRRK1, LRRK2, DYRK1A, MST1 and TBK1 for their capability of phosphorylating Rab GTPases. We identified several novel kinase:Rab pairs, such as LRRK1:Rab43 and TBK1:Rab29. Further, we comprehensively assessed what makes a Rab GTPase a good kinase substrate, considering the Rab nucleotide-binding state and the Rab primary sequence. In a systematic mutational study, Rab variants with modulated phosphorylation properties were established, leading to the identification of a LRRK2 recognition patch in the Rab α3 helix. A Glu to Arg exchange in that patch increased the phosphorylation 18-fold, indicating that Rabs are suboptimal LRRK2 substrates. Given that this effect is also observed in a cellular model, we propose that our variants will be excellent tools for analysing the physiological function of Rab phosphorylation.

Pseudomonas aeruginosa PA01 is one of the major causes of disease persistence and mortality in patients with lung pathologies, relying on various host metabolites as carbon and energy sources for growth. The ict-ich-ccl operon (pa0878, pa0882 and pa0883) in PAO1 is required for growth on the host molecule itaconate, a C5-dicarboxylate. However, it is not known how itaconate is taken up into P. aeruginosa. Here, we demonstrate that a genetically linked tripartite ATP-independent periplasmic (TRAP) transporter (pa0884-pa0886), which is homologous to the known C4-dicarboxylate-binding TRAP system, is essential for growth on itaconate, but not for the closely related C4-dicarboxylate succinate. Using tryptophan fluorescence spectroscopy, we demonstrate that the substrate-binding protein (SBP), IctP (PA0884), binds itaconate but still retains higher affinity for the related C4-dicarboxylates. The structures of IctP bound to itaconate (1.80 Å) and succinate (1.75 Å) revealed an enclosed ligand-binding pocket with ion pairing interactions with the ligand carboxylates. The C2 methylene group that is the distinguishing feature of itaconate compared with succinate is accommodated by a unique change in the IctP-binding site from a Leu to Val, which distinguishes it from closely related C4-dicarboxylate-binding SBPs. Together, these data suggest that this transporter, which we name IctPQM, has duplicated from a canonical C4-dicarboxylate transporter, and its evolution towards itaconate specificity enables this pathogen to now access a key metabolite for persistence in the host.

Mechanistic studies of biomolecular machines involved in intracellular protein degradation-such as the caseinolytic protease P, ATPases associated with diverse cellular activities (AAA+) motors, and the high-temperature requirement A family of enzymes-are of great interest as they are implicated in a host of human diseases. The function of these systems is dependent on both their fine-tuned three-dimensional structure and the conformational dynamics that modulate this structure. Their large sizes, inherent conformational plasticity, and oligomeric heterogeneity dictate that their mechanism of action cannot be deciphered by any one method. Synergistic application of methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), nuclear magnetic resonance (NMR), and single-particle electron cryomicroscopy (cryo-EM) has uniquely positioned researchers to tackle the outstanding questions in this area of structural biology. Cryo-EM enables structural characterization and modeling of the large and conformationally heterogeneous complexes involved in protein degradation, while methyl-TROSY NMR enables monitoring structural transitions and conformational dynamics of these systems in response to various stimuli in solution at atomic resolution. This review highlights how combining these two approaches offers a distinct and powerful means to unravel allosteric pathways within complex, multipartite biomolecular machines.

Intrabodies are intracellularly expressed high-affinity protein binders such as nanobodies and monobodies that offer an alternative approach to small molecules. However, the maturation of intrabody technology into new therapeutic modalities has been limited by the availability of a clinically relevant delivery system enabling sufficiently high levels of protein to be expressed in the cytosol. Here, we use lipid nanoparticle (LNP) systems based on clinically approved formulations for the efficient intracellular delivery of mRNAs encoding for intrabodies targeting mixed lineage kinase domain-like pseudokinase (MLKL) and apoptosis-associated speck-like protein containing a CARD (ASC), key mediators of the necrotic cell death modalities, necroptosis and pyroptosis, respectively. LNP delivery of intrabody mRNA resulted in robust protein expression, with an MLKL-binding intrabody preventing MLKL membrane translocation and protecting against necroptotic cell death. Similarly, LNP delivery of a bivalent intrabody targeting the inflammasome adaptor protein ASC protected against NLRP3 and AIM2 inflammasome-driven responses, including caspase-1 and IL-1β activation and gasdermin D-driven pyroptotic killing. These findings establish that LNPs harbouring anti-necrotic intrabody mRNAs allow for sufficient intracellular expression to neutralize necrotic cell death signalling and provide a general, clinically relevant, strategy for delivering therapeutic intrabodies into cells.

Mitochondria are multifaceted organelles that support numerous cellular metabolic pathways, including the biosynthesis of nucleotides required for cell growth and proliferation. Owing to an ancient endosymbiotic origin, mitochondria contain multiple copies of their own genome and therefore demand sufficient (deoxy)nucleotides in the mitochondrial matrix for DNA replication and transcription into RNA. Disturbed mitochondrial deoxynucleotide homeostasis can lead to a decline in mitochondrial DNA abundance and integrity, causing mitochondrial diseases with diverse and severe symptoms. Mitochondrial nucleotides are not only required for nucleic acid synthesis but also for bioenergetics and mitochondrial enzymatic activity. This review first explores how mitochondria supply energy and anabolic precursors for nucleotide synthesis and how the mitochondrial network influences the spatial control of cellular nucleotide metabolism. Then follows an in-depth discussion of the mechanisms that supply mitochondria with sufficient and balanced nucleotides and why these mechanisms are relevant to human mitochondrial disease. Lastly, the review highlights the emergence of regulated mitochondrial nucleotide supply in physiological processes including innate immunity and discusses the implications of dysregulated mitochondrial and cytosolic nucleotide homeostasis in pathophysiology.

Human kynurenine aminotransferase 1 (hKYAT1) plays a crucial role in the transamination of aromatic amino acids and kynurenine. This promiscuous homodimeric enzyme transaminates various amino acids into their corresponding α-keto acids. Additionally, hKYAT1 is known to catalyze the β-elimination of cysteine-S conjugates and cysteine-Se conjugates. In this study, we performed mutational analyses of hKYAT1, targeting its catalytic, ligand-binding, and substrate-binding sites. The transamination activity of 13 mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the β-elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substratestabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and β-elimination of MSC by 2- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398, F125, and N185 substantially reduced MSC transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of l-kynurenine, a natural substrate of hKYAT1. Altogether, these findings support future investigation into hKYAT1 as a modifiable target in selenium-mediated anticancer approaches.

Mitochondria are multifaceted organelles that support numerous cellular metabolic pathways, including the biosynthesis of nucleotides required for cell growth and proliferation. Owing to an ancient endosymbiotic origin, mitochondria contain multiple copies of their own genome and therefore demand sufficient (deoxy)nucleotides in the mitochondrial matrix for DNA replication and transcription into RNA. Disturbed mitochondrial deoxynucleotide homeostasis can lead to a decline in mitochondrial DNA abundance and integrity, causing mitochondrial diseases with diverse and severe symptoms. Mitochondrial nucleotides are not only required for nucleic acid synthesis but also for bioenergetics and mitochondrial enzymatic activity. This review first explores how mitochondria supply energy and anabolic precursors for nucleotide synthesis and how the mitochondrial network influences the spatial control of cellular nucleotide metabolism. Then follows an in-depth discussion of the mechanisms that supply mitochondria with sufficient and balanced nucleotides and why these mechanisms are relevant to human mitochondrial disease. Lastly, the review highlights the emergence of regulated mitochondrial nucleotide supply in physiological processes including innate immunity and discusses the implications of dysregulated mitochondrial and cytosolic nucleotide homeostasis in pathophysiology.