Biochemistry Biochemistry最新文献

Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining, we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for protein stability. Cysimiditides bind a Zn²⁺ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn²⁺ is required for the methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn²⁺-tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn²⁺-tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally confirmed RiPPs harboring aspartimides and represent the first RiPP class that has an obligate metal ion.

Dimethyladenosine transferase 1 (DIMT1) is an RNA N^6,6-dimethyladenosine (m₂^6,6A) methyltransferase. DIMT1’s role in pre-rRNA processing and ribosome biogenesis is critical for cell proliferation. Here, we investigated the minimal number of residues in a positively charged cleft on DIMT1 required for cell proliferation. We demonstrate that a minimum of four residues in the positively charged cleft must be mutated to alter DIMT1’s RNA-binding ability. The variant (4mutA-DIMT1), which presents reduced RNA binding affinity, is diffuse in the nucleoplasm and nucleolus, in contrast with the primarily nucleolar localization of wild-type DIMT1. The aberrant cellular localization significantly impaired 4mutA-DIMT1’s role in supporting cell proliferation, as shown in competition-based cell proliferation assays. These results identify the minimum region in DIMT1 to target for cell proliferation regulation.

The apoptosome, a critical protein complex in apoptosis regulation, relies on intricate interactions between its components, particularly the proteins containing the Caspase Activation and Recruitment Domain (CARD). This work presents a thorough computational analysis of the stability and specificity of CARD–CARD interactions within the apoptosome. Departing from available crystal structures, we identify important residues for the interaction between the CARD domains of Apaf-1 and Caspase-9. Our results underscore the essential role of these residues in apoptosome activity, offering prospects for targeted intervention strategies. Available experimental complex structures were able to validate the protein–protein docking consensus approach used herein. We furthermore extended our analysis to explore the specificity of CARD–CARD interactions by cross-docking experiments between apoptosome and PIDDosome components, between which there should not be any interaction despite belonging to the same death fold subfamily. Our findings indicate that native interactions within individual complexes exhibit greater stability than the cross-docked complexes, emphasizing the specificity required for effective protein complex formation. This study enhances our understanding of apoptotic regulation and demonstrates the utility of computational approaches in elucidating intricate protein–protein interactions.

Aggregation of α-synuclein (α-Syn) and Lewy body (LB) formation are the key pathological events implicated in Parkinson’s disease (PD) that spread in a prion-like manner. However, biophysical and structural characteristics of toxic α-Syn species and molecular events that drive early events in the propagation of α-Syn amyloids in a prion-like manner remain elusive. We used a neuronal cell model to demonstrate the size-dependent native biological activities of α-Syn fibril seeds. Biophysical characterization of the fibril seeds generated by controlled fragmentation indicated that increased fragmentation leads to a reduction in fibril size, correlating directly with the extent of fragmentation events. Although the size-based complexity of amyloid fibrils modulates their biological activities and fibril amplification pathways, it remains unclear how the variability of fibril seed size dictates its specific uptake mechanism into the cells. The present study elucidates the mechanism of α-Syn fibril internalization and how it is regulated by the size of fibril seeds. Further, we demonstrate that size-dependent endocytic pathways (dynamin-dependent clathrin/caveolin-mediated) are more prominent for the differential uptake of short fibril seeds compared to their longer counterparts. This size-dependent preference might contribute to the enhanced uptake and transcellular propagation of short α-Syn fibril seeds in a prion-like manner. Overall, the present study suggests that the physical dimension of α-Syn amyloid fibril seeds significantly influences their cellular uptake and pathological responses in the initiation and progression of PD.

Metal ions are essential for all life. In microbial cells, potassium (K⁺) is the most abundant cation and plays a key role in maintaining osmotic balance. Magnesium (Mg²⁺) is the dominant divalent cation and is required for nucleic acid structure and as an enzyme cofactor. Microbes typically require the transition metals manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn), although the precise set of metal ions needed to sustain life is variable. Intracellular metal pools can be conceptualized as a chemically complex mixture of rapidly exchanging (labile) ions, complemented by those reservoirs that exchange slowly relative to cell metabolism (sequestered). Labile metal pools are buffered by transient interactions with anionic metabolites and macromolecules, with the ribosome playing a major role. Sequestered metal pools include many metalloproteins, cofactors, and storage depots, with some pools redeployed upon metal depletion. Here, I review the size, composition, and dynamics of intracellular metal pools and highlight the major gaps in understanding.

The mitochondrial outer membrane (OMM) β-barrel proteins link the mitochondrion with the cytosol, endoplasmic reticulum, and other cellular membranes, establishing cellular homeostasis. Their active insertion and assembly in the outer mitochondrial membrane is achieved in an energy-independent yet highly effective manner by the Sorting and Assembly Machinery (SAM) of the OMM. The core SAM constituent is the 16-stranded transmembrane β-barrel Sam50. For over two decades, the primary role of Sam50 has been linked to its function as a chaperone in the OMM, wherein it assembles all β-barrels through a lateral gating and β-barrel switching mechanism. Interestingly, recent studies have demonstrated that despite its low copy number, Sam50 performs various diverse functions beyond assembling β-barrels. This includes maintaining cristae morphology, bidirectional lipid shuttling between the ER and mitochondrial inner membrane, import of select proteins, regulation of PINK1-Parkin function, and timed trigger of cell death. Given these multifaceted critical regulatory functions of SAM across all eukaryotes, we now reason that SAM merely moonlights as the hub for β-barrel biogenesis and has indeed evolved a diverse array of primary roles in maintaining mitochondrial function and cellular homeostasis.

Traumatic brain injury (TBI) is a serious health condition that affects an increasing number of people, especially veterans and athletes. TBI causes serious consequences because of its long-lasting impact on the brain and its alarming frequency of occurrence. Although the brain has some natural protective mechanisms, the processes that trigger them are poorly understood. Fibroblast growth factor (FGF) proteins interact with receptor proteins to protect cells. Gaps in the literature include how basic-FGF (bFGF) is activated by heparin, can heparin-like molecules induce neural protection, and the effect of allosteric binding on bFGF activity. To fill the gap in our understanding, we applied temperature replica exchange to study the influence of heparin binding to bFGF and how mutations in bFGF influence stability. A new favorable binding site was identified by comparing free energies computed from the potential of mean force (PMF). Although the varied sugars studied resulted in different interactions with bFGF compared to heparin, they each produced structural effects similar to those of bFGF that likely facilitate receptor binding and signaling. Our results also demonstrate how point mutations can trigger the same conformational change that is believed to promote favorable interactions with the receptor. A deeper atomic-level understanding of how chemicals are released during TBI is needed to improve the development of new treatments for TBI and could contribute to a better understanding of other diseases.

Streptomyces griseolus CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs. We successfully crystallized complexes of wild-type CYP105A1 (WT) and the R84A variant with diclofenac (DIF) or flufenamic acid (FLF). In the WT, the carboxyl group of DIF formed a charged hydrogen bond with Arg84. In contrast, in R84A, the carboxyl group formed two bidentate charged hydrogen bonds with Arg73. The C4′ atom of the benzene ring of DIF, which undergoes hydroxylation by WT and R84A, was positioned approximately 4 Å from the heme iron. Binding of FLF was nearly the same in both WT and R84A. The carboxyl group of FLF formed charged hydrogen bonds with Arg73. In both WT and R84A, FLF appeared to be fixed by this charged hydrogen bonding with Arg73 during the reaction, and the C4′ atom, which undergoes hydroxylation, must face the heme iron. Thus, the dihedral angles of the two N–C bonds connecting the two benzene rings of FLF needed to rotate by 78° and −71°, respectively. The temperature factors of the F-G loop, helix F, and helix G of R84A were remarkably higher than those of WT. This suggests that these regions in R84A are much more flexible compared to those of WT, which may consequently affect substrate binding and product release.

DtpC was isolated from the ditryptophenaline biosynthetic pathway found in filamentous fungi as a cytochrome P450 (P450) that catalyzes the dimerization of diketopiperazines. More recently, several similar P450s were discovered. While a vast majority of such P450s generate asymmetric diketopiperazine dimers, DtpC and other fungal P450s predominantly catalyze the formation of symmetric dimer products. Dimeric compounds can have interesting biological activities, and the mode of dimerization can substantially affect their bioactivities substantially. Here, we set out to examine the mechanism and scope of diketopiperazine dimerization catalyzed by DtpC using both chemically modified substrate molecules and DtpC mutants that were selected by the screening of randomly mutated recombinant variants. Use of N1- and N10-methylated diketopiperazine substrates supports the proposal that the initial radical formation occurs by extraction of the N1 indole nitrogen for this fungal P450 dimerase. Further in vitro studies revealed that DtpC was capable of accepting a range of structurally variable substrates, including N-demethylated diketopiperazines, and forming symmetric homo- and heterodimeric products. Moreover, the introduction of single mutations identified through the screening of random mutants at and around the substrate-binding pocket led to the conversion of DtpC into a catalyst that predominantly generated asymmetric dimers of various diketopiperazines. The versatility of DtpC can serve as a good starting point for directed evolution of P450s that can serve as versatile catalysts for generation of various dimers of not only diketopiperazines derived from standard and nonstandard amino acids but also possibly structurally more divergent analogs of diketopiperazines.

Heart muscle systolic and diastolic function is controlled on a beat-to-beat basis by the calcium-dependent activation of the contractile myofilaments but modulated by neurohumoral signaling pathways coupled to the activation of intracellular effector molecules such as protein kinases. Phosphorylation of myofilament regulatory proteins such as cardiac troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) has important regulatory function for the heart by controlling both cardiac inotropy and lusitropy. Sympathetic signaling activates both α- and β-adrenergic receptors on the surface of cardiomyocytes, which leads to an increase in cMyBP-C phosphorylation via protein kinase C (PKC)/D (PKD) and protein kinase A (PKA) signaling, respectively. However, the functional interactions between the PKC/PKD and PKA phosphorylation sites on cMyBP-C have remained uncharacterized. Here, using a combination of site-specific phosphorylation of recombinant N-terminal domains of cMyBP-C and in situ functional assays, we show that the PKC/PKD and PKA phosphorylation sites have antagonistic effects on myofilament activation. PKA phosphorylation on multiple sites in the N-terminal domains of cMyBP-C reduces both its activating and inhibiting effect on myofilament activation in the absence and presence of activator Ca²⁺, respectively. In contrast, PKC phosphorylation increases myofilament activation and blunts the inhibitory effect of PKA phosphorylation. Our results lead to a new model of phosphoregulation of cMyBP-C with important implications for both health and disease states of the heart.