Biochemistry Biochemistry最新文献

The activity of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 1 (SERCA1) of muscle is inhibited by small ankyrin1 (sAnk1), a ∼17 kDa transmembrane protein that stabilizes the network compartment of the sarcoplasmic reticulum (SR). sAnk1 binds to sarcolipin (SLN), a 31 amino acid peptide inhibitor of SERCA1, to ablate its inhibitory activity. SERCA1 is also inhibited by phospholamban (PLN), which shares homology with both sAnk1 and SLN. Here we use cotransfection of COS7 cells, coimmunoprecipitation and bimolecular fluorescent complementation (BiFC) to show that sAnk1 associates with PLN and forms a 3-way complex with PLN and SERCA1. Anisotropy-based FRET (AFRET) studies of Cerulean-SERCA1 with Venus-tagged sAnk1 and PLN confirmed the presence of a 3-way complex. ATPase assays showed that, unlike its effects on SLN, sAnk1 does not ablate PLN’s inhibition of SERCA1 activity. Our results are consistent with a model in which, in forming a three-way complex, PLN binds to SERCA1 first, followed by binding to sAnk1. This modeling suggests that the interactions of PLN, SLN and sAnk1 with SERCA1, either alone or in pairs, are distinct and have different effects on SERCA1’s enzymatic activity.

Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from Leishmania major (LmGAPDH) is present in extracellular vesicles and inhibits host TNF-α expression during infection via post-transcriptional repression. LmGAPDH binding to AU-rich elements in the 3′-untranslated region of TNF-α mRNA (TNF-α ARE) was sufficient for limiting cytokine production, but the TNF-α ARE binding residues in LmGAPDH remain unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurements revealed that the inhibition by the TNF-α ARE was competitive with respect to the cofactor NAD⁺ in LmGAPDH. To identify the TNF-α ARE binding residues of LmGAPDH, we performed a systematic mutational analysis of its NAD⁺ binding domain. Catalytic activity measurements indicated that both R13 and N336 amino acids in the NAD⁺ binding site are absolutely required for activity, whereas other mutants, including I14A, R16A, D39A, and T112A, showed higher K_m (lower affinity) values for NAD⁺ binding and lower catalytic activity. REMSA studies revealed that the replacement of Arg13 with Ala/Lys or Asn336 with Ala resulted in a complete loss of binding to the TNF-α ARE. I14A, R16A, D39A, and T112A residues at or near the NAD⁺ binding site showed lower binding to the TNF-α ARE compared to the wild-type protein. Protein-induced fluorescence enhancement (PIFE) studies and in vitro protein translation assays further confirmed the REMSA results. Based on our findings, the NAD⁺ binding residues in LmGAPDH are important for TNF-α ARE binding.

Peptidylarginine deiminases (PADs) catalyze the calcium-dependent conversion of arginine to citrulline, which affects diverse cellular processes. Among the human PAD isoforms, PAD2 and PAD4 are particularly relevant because of their distinct tissue distributions and substrate preferences. However, the lack of isoform-selective substrates has limited our ability to discriminate between their activities in biological systems. In this study, we developed PAD2- and PAD4-selective fluorogenic peptide substrates using the Hybrid Combinatorial Substrate Library (HyCoSuL) strategy, which incorporates both natural and over 100 unnatural amino acids. Substrate specificity profiling at P4–P2 positions revealed that PAD2 tolerates a broader range of residues, particularly at the P2 position, whereas PAD4 displays more selective preferences, favoring aspartic acid at this site. Based on these insights, we designed and validated peptide substrates with high selectivity for PAD2 or PAD4, enabling isoform-specific kinetic analysis in vitro. We demonstrated the utility of these substrates in profiling PAD activity in THP-1 macrophages, revealing dominant PAD2 activity in PMA (phorbol 12-myristate 13-acetate)/LPS (lipopolysaccharide)-stimulated monocytes. Furthermore, PAD4-mediated citrullination of vimentin modulates its susceptibility to caspase and calpain cleavage, potentially altering its function as a damage-associated molecular pattern (DAMP). Our findings provide a framework for the development of PAD-selective inhibitors and chemical probes, enabling the precise dissection of isozyme-specific PAD functions in health and disease.

Dynamin family proteins typically do not depend on higher-order oligomerization; instead, dimerization and/or tetramerization is sufficient for their target membrane recruitment. Here, we demonstrate that dimerization/tetramerization alone is not enough, but self-assembly into a higher-order structure is also required for targeting a dynamin-related protein, dynamin-related protein 6 (Drp6), to the nuclear membrane. We identify residues 411-GKFR-414 as important for higher-order oligomerization of Drp6 but dispensable for its dimerization/tetramerization. Furthermore, while the mutation of GKFR residues does not affect membrane-binding ability in vitro, it inhibits the nuclear localization of Drp6 in vivo. Ultrastructure expansion microscopy and fast super-resolution live cell imaging demonstrate that the cytosolic, higher-order self-assembled structure of Drp6 is recruited to the nuclear envelope. These findings establish self-assembly into a higher-order oligomer as a prerequisite for target membrane recruitment of a dynamin-related protein.

Alzheimer’s disease is a neurodegenerative disease whose pathological hallmark is the fibrilization of the amyloid-β (Aβ) peptides. Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), inhibit the Aβ aggregation in vitro; however, the molecular basis for the inhibition remained unclear. In this study, we analyzed the interactions of n-3 PUFAs with the partial peptides of 42-residue Aβ via molecular simulations. The analysis predicted that DHA and EPA were preferred over their derivatives in terms of the calculated free energy changes of the ligand–Aβ binding. The results of our simulations were validated using experimental methods, and the structural characteristics identified in in silico analysis were also confirmed to be important elements inin vitro experiments. This study enabled a mechanistic understanding of n-3 PUFAs to protect against Aβ fibril formation and offer a molecular basis for designing therapeutics against Alzheimer’s disease.

Chromatin remodelers maintain the chromatin structure and hence gene expression. Imitation SWItch, ISWI, is a chromatin remodeler, which regulates nucleosome spacing across the genome by its adenosine 5′-triphosphate (ATP)-dependent nucleosome sliding activity. To understand how this happens requires identification of the conformational changes that occur in all domains of ISWI during the entire nucleosome sliding cycle. Using the hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) methodology, we have monitored the conformational dynamics of Drosophila FL-ISWI at all the stages of nucleosome sliding. Our data show that, in the resting state, FL-ISWI is intrinsically dynamic in many regions, including the N- and C-terminal regulatory regions. During nucleosome sliding, different regions of the ATPase domain, which bind to the nucleosomal DNA, undergo a major conformational change, and the C-terminal HSS domain switches from a stable state to a more dynamic state. ISWI adopts distinct conformations in its nucleosome bound and sliding states as the interactions established by it upon binding to the nucleosome are broken during DNA translocation. HDX-MS has made it possible to characterize multiscale dynamics from small fluctuations to large structural changes occurring in all the domains of FL-ISWI during the different steps of nucleosome sliding. The structural mechanism revealed for ISWI has implications for several other protein families containing a Rec-A domain ATPase core.

G protein-coupled receptors (GPCRs) are central to cellular signaling and therapeutic targeting. Ligands that activate the same GPCR can selectively activate some signaling pathways over others, a phenomenon termed biased agonism. Additionally, the same ligand and receptor complex can elicit distinct signaling profiles in different subcellular locations (location bias). Here, we examine how various biased agonists influence the recruitment of β-arrestins 1 and 2 induced by the angiotensin II type 1 receptor at the receptor, plasma membrane, and early endosomes. We also assessed β-arrestin conformational states at the receptor and plasma membrane. Using split luciferase and BRET assays, we demonstrate that angiotensin II, its G protein-biased analogs (TRV055, TRV056), and its β-arrestin-biased analogs (TRV023, TRV026, TRV027, TRV034) functionally stratify into two clusters. G protein-biased agonists and AngII predominantly favor a receptor−β-arrestin core complex conformation driven by engagement of the β-arrestin finger loop with the receptor core. In contrast, β-arrestin-biased agonists promote a tail complex configuration of receptor-associated β-arrestins. However, the conformations of β-arrestins monitored at the plasma membrane were found to be unaffected by ligand bias. Furthermore, balanced and G protein-biased ligands induced higher levels of ERK activation in subcellular locations (nucleus, cytosol, and early endosomes) over the β-arrestin-biased ligands, but equal ERK activity at the plasma membrane. Our findings highlight the interplay between ligand and location biases in dictating GPCR signaling, revealing new insights into the molecular mechanisms driving selective signal propagation.

3′-Phosphoadenosine-5′-phosphosulfate (PAPS), a universal sulfate donor for sulfation reactions, is indispensable for synthesizing bioactive molecules including therapeutic glycosaminoglycans and sulfolipids; however, its enzymatic production on an industrial scale is constrained by ATP overconsumption and the limited free enzyme reusability. We report an integrated biocatalytic platform combining ATP regeneration with affinity immobilization to enable sustainable PAPS biosynthesis. A polyphosphate kinase-driven ATP regeneration system achieved 86% PAPS conversion efficiency by regenerating ADP using low-cost polyphosphate. Biotin–streptavidin affinity immobilization enhanced operational stability, retaining >50% activity over six reuse cycles with a cumulative PAPS titer of 12.02 g/L. Coupling adenosine-converting Saccharomyces cerevisiae whole-cell catalysts with this system decreased substrate costs by 80.7% and delivered 96% molar PAPS yield from adenosine. This work provides a sustainable platform for industrial PAPS biosynthesis to promote sulfated biomolecule production, including glycosaminoglycans and other therapeutics.

Enzymes are involved in the biosynthesis of a variety of secondary metabolites found in nature. The catalytic mechanism is regulated by the three-dimensional structure of the enzyme, particularly at the catalytic site, resulting in the synthesis of natural products with complex conformations derived from a regioselective, chemoselective, or stereoselective preference of the enzyme reaction. Prenyltransferase, which belongs to the prenylsynthase superfamily, catalyzes the condensation of isoprene to an aromatic compound, consequently producing a terpenoid scaffold structure. Prenyltransferase thus plays an important role in expanding the chemical diversity of the terpenoids. Although the three-dimensional structures of prenylsynthases categorized in the same superfamily have been resolved, the catalytic mechanism of prenyltransferase has been veiled. In this study, we determined the X-ray crystal structure of a novel prenyltransferase, Ord1, which is derived from Streptomyces. Here, we report the enzymatic characteristics of the Ord1 and discuss its catalytic mechanism.

Transcription by RNA polymerase (RNAP) lies at the heart of gene expression in all organisms. The speed with which RNAPs produce RNA is tuned, in part, by signals in the transcribed nucleic acid sequences, which temporarily arrange RNAPs into a paused conformation that is unable to extend the RNA. In turn, the altered transcription kinetics of paused RNAPs determine the three-dimensional shape into which RNA ultimately folds and promote or inhibit cotranscriptional events. While pause sequence determinants have been characterized for multisubunit RNAPs in bacteria and in eukaryotic nuclei, this information is lacking for the single-subunit, T-odd phage-like RNAP of human mitochondria, POLRMT. Here, we developed a robust nucleic acid scaffold system to reconstitute POLRMT transcription in vitro and identified multiple transcriptional pause sites on the human mitochondrial DNA (mtDNA). Using one of the pause sequences as a representative, we performed a suite of mutational studies to pinpoint the nucleic acid elements that enhance, weaken, or completely abolish POLRMT pausing. Based on these mutational results, we constructed a consensus pause motif expected to cause strong pausing for POLRMT: 5′-R_–10NNNNNNNGT_–1G₊₁-3′, where −1 is the 3′ nascent RNA nucleotide in the POLRMT active site, +1 is the incoming NTP to be added to the nascent RNA, R is A or G, and N is any base. Strikingly, most of the consensus pause elements in this motif are the same for multisubunit prokaryotic and nuclear RNAPs, hinting at potentially shared features of the pausing mechanism despite the structural differences between polymerases. Finally, a search of the human mtDNA for this pause motif revealed multiple predicted pause sites with potential roles in mitochondrial cotranscriptional processes.