Biochemistry Biochemistry最新文献

Antimicrobial resistance (AMR) is a global healthcare emergency, directly causing 1.3 million deaths per year and predicted to increase dramatically over the coming decades. Understanding the molecular mechanisms underpinning antibiotic resistance is central to approaches for AMR surveillance and diagnosis in a clinical laboratory. Current antibiotic susceptibility tests are designed to detect canonical mechanisms of AMR that are functional on standard laboratory media. However, increasing evidence suggests that host and environmental factors can influence antibiotic susceptibility. In this perspective, we review known condition-dependent mechanisms of AMR and define them into four mechanistic classes: (1) Regulation of canonical AMR mechanisms by the host environment; (2) Changes to cellular respiration; (3) Increased metabolic capability; and (4) Metabolic control of tolerance and persistence. We further explore how these noncanonical AMR mechanisms can impact antibiotic susceptibility test results, and how increased mechanistic understanding might be used to optimize antibiotic therapy.

Peroxiredoxins (Prdxs) are thiol proteins that function both as antioxidants and as regulators of cell signaling. This article focuses on the biological chemistry of mammalian typical 2-Cys Prdxs and how their molecular properties relate to cellular function. These Prdxs operate through a complex mechanism that involves redox changes at two active sites, coupled to conformational changes and reversible oligomerization. The reduced forms react extremely rapidly with H₂O₂ but, paradoxically for efficient antioxidants, condensation of the resultant sulfenic acid to the intramolecular disulfide is remarkably slow. Consequently, turnover plateaus as the H₂O₂ concentration rises and further slows as oxidized thioredoxin accumulates and recycling becomes limited. Therefore, these Prdxs are potent scavengers of H₂O₂ at low concentrations but are less well equipped for high fluxes. The biochemical properties of Prdxs are well suited for sensing H₂O₂ and relaying oxidation to less reactive thiol proteins in redox-regulated signaling pathways. Several relays have been well characterized, but it is proving challenging to establish whether this is a widespread signaling mechanism in mammalian cells. An alternative signaling mechanism is for Prdxs to act as negative regulators. This requires the direct oxidation of signaling proteins that bypasses the efficient reaction of H₂O₂ with Prdxs. Various mechanisms have been proposed, but most remain speculative. Overall, although we have detailed knowledge of the properties of mammalian Prdxs and evidence that they perform important cell functions, there are still major mechanistic gaps to bridge between the two.

Synucleinopathies are neurodegenerative disorders marked by the accumulation of misfolded α-synuclein and its familial mutants in brain cells. The mechanistic understanding of how α-synuclein mutations exacerbate disease remains unsolved. Here, the in vitro aggregation kinetics of α-synuclein and its mutants revealed that A30P and a recently discovered A30G variant displayed aggregation kinetics uncharacteristically slower than those of wild-type (WT) and other mutants. We delineated the amyloidogenesis pathway of these variants by characterizing different intermediates through time-dependent CD and AFM analysis. AFM-Raman spectroscopy and proteinase-K digestion further distinguished structural features of these species. We infer that WT and the A30 variants aggregate through a common pathway, albeit with variant-specific rates, yielding kinetically metastable aggregates with structural differences in the order A30P < A30G < WT. These metastable aggregates underwent further rearrangements to form stable fibrils upon vigorous agitation, incubation with osmolytes, or seeding with fibrillar seeds. Addition of the amyloid-modifying compound EGCG to the metastable and the final fibrillar states converted them to β-sheet-rich small fibrillar and prefibrillar structures, respectively. However, EGCG directed monomers toward amorphous aggregation. We present a viewpoint that A30 mutations that are located outside the canonical amyloid core of α-synuclein could augment its toxic gain of function by producing kinetically long-lived prefibrillar species rather than by promoting thermodynamically alternative conformations. These kinetically metastable conformers of varying hierarchy were differentially sensitive to EGCG. Our findings provide significant insights into how α-synuclein mutations contribute to synucleinopathies and how amyloid modulators differentially act on intermediates versus mature fibrils.

β-Lactamases are the major mechanism of antimicrobial resistance to β-lactam antibiotics in Gram-negative bacteria. Continuous exposure to these drugs has resulted in >12,400 known variants due to evolutionary pressure. These enzymes represent a unique model for studying protein evolution, as bacterial survival depends on expressing β-lactamases that inactivate the specific β-lactam antibiotic that the bacterium encounters. In this perspective, we discuss salient examples in which changes in protein dynamics have played a role in the evolution of β-lactamases. Our analysis is based in the concept that proteins explore alternative conformations, and mutations can favor some of these conformations, providing a “gain-of-function” to the enzyme. The role of less-populated conformations and cryptic binding sites in evolution is discussed, as well as state-of-the-art experimental approaches that can study the role of alternative conformations in evolution. This approach also reveals opportunities to develop allosteric inhibitors that take advantage of these alternative conformations.

Lipids are fundamental to life, serving not only as membrane components but also as key players in signaling, energy storage, and cellular identity. While the well-known “lipid divide” between Bacteria and Archaea marks a major early evolutionary step, recent findings suggest that lipid-based divergences extend well beyond prokaryotic domains. In this Perspective, we highlight multiple lipid divergences that coincide with key phylogenetic splits, including the unikont-bikont or Opimoda-Diphoda separation, the Amoebozoa-Opisthokonta (Obazoa) and fungi-metazoa splits, the invertebrate-vertebrate transition, and even intragenus divergence. These divergences are often reflected in the lineage-specific presence or absence of lipid biosynthetic enzymes, regulatory proteins, and functional effectors. By mapping these molecular players alongside the lipid class distribution, we propose an expanded model of the lipid divide as a recurring hallmark of evolution. We believe this lipid-centric view will offer fresh insights into the molecular basis of cellular complexity and evolutionary innovation and encourage further investigation into additional, yet-unrecognized lipid divergence events.

Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a critical role in NO-based regulation of cardiovascular and pulmonary function. The physiological importance of sGC signaling has led to the development of drugs, known as stimulators and activators, which increase sGC catalytic function. Here we characterize a newly developed stimulator, CYR715, which is a particularly potent stimulator of Manduca sexta (Ms) sGC catalytic function even in the absence of NO, increasing activity of the NO-free enzyme to 45% of full catalytic activity. CYR715 also increased the catalytic activity of Ms sGC βC122A and βC122S variants, with a marked stimulation of the NO-free βC122S variant to 74% of maximum. High-resolution cryo-electron microscopy structures were solved for CYR715 bound to Ms sGC βC122S revealing that CYR715 occupies the same binding site as the characterized sGC stimulators YC-1 and riociguat. Additionally, the core scaffold of CYR715 makes a binding interaction with βC78 while the flexible tail can interact with αR429 or βY7 and E361. Conformational extension of sGC following NO, YC-1, or CYR715 binding was characterized using small-angle X-ray scattering, revealing that while ligand binding results in sGC extension this extension does not directly correlate to observed activity. This suggests that not all conformational extensions of sGC result in increased catalytic activity, and that effective stimulators assist in converting extension into catalytic function.

The brain is the most cholesterol-rich organ in the body, and ApoE is the main lipid carrier protein in the brain. Although very little, if any, ApoE exists in its apoprotein form in physiological fluids, recombinant ApoE is typically prepared in a lipid-free state to study its physiological functions. We describe a lipid nanoparticle (LNP) form of ApoE as a primary extracellular product of the eukaryotic protein export system. Whereas the apoprotein is the dominant secreted product when the APOE gene is overexpressed in mammalian cells, an LNP form of ApoE is also observed. The LNP form is, however, the major secreted product from unmodified CCF-STTG1 astrocytoma cells. The C-terminal domain of ApoE plays a key role in LNP biosynthesis as the ApoE3 W210* truncation mutant is secreted without lipidation. Secreted ApoE LNPs are markedly better substrates than the apoprotein itself for further growth via the action of ATP-dependent lipid pumps. Compared to ApoE3 or the Alzheimer’s disease-protective ApoE2 variant, the recovered yield of the LNP form of the disease-predisposing ApoE4 variant is higher. Intriguingly, the LNP yield of the rare disease-protective R251G variant of ApoE4 is comparable to that of ApoE3 and ApoE2. Analogous to the well-documented intracellular biosynthesis of ApoB-containing LNPs, the biogenesis and pathophysiological relevance of the LNP form of ApoE warrant further investigation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the most conserved proteins across all kingdoms of life, has a multitude of moonlighting functions beyond its enzymatic role in glycolysis. Metal binding to GAPDH has previously been reported to inhibit enzymatic activity in several prokaryotic and eukaryotic systems, although the mechanism of inhibition has not been elucidated. In this study, we examined the effects of zinc, silver, and copper ions on Escherichia coli GAPDH (ecGAPDH) and explore the mechanism of inhibition via enzymatic activity assays under aerobic and anaerobic conditions, electron paramagnetic spectroscopy, and mass spectrometry. This study shows that Zn²⁺ does not affect ecGAPDH activity, while Cu²⁺ causes redox inactivation that oxidizes the protein upon reduction to Cu⁺. Cu⁺ binds tightly to the protein (log K_a = 15.2 ± 0.2, pH 7.4), with diminished affinity in the presence of G3P substrate. Although the anaerobic binding of Cu⁺ or Ag⁺ moderately diminishes catalytic turnover, these ions sensitize the protein to rapid and complete oxidative inactivation in the presence of oxygen. Oxidative modification of the active site cysteine, including glutathionylation, is reversible. This oxidative process, which occurs upon exposure to Cu and Ag, bestows GAPDH the ability to act as an all-purpose redox switch responsive to toxic metals as well as reactive oxygen species. This work provides insight into shared mechanisms by which cells use redox inactivation of sentinel enzymes like GAPDH to redirect metabolic processes for cellular protection.

Cytochrome P450 121A1 (CYP121A1) of Mycobacterium tuberculosis is involved in the essential synthesis of mycocyclosin from its substrate, dicyclotyrosine (cYY). As such, CYP121A1 continues to garner significant interest as a drug target. In this study, all-atom molecular dynamics simulations have been employed to investigate the behavior of bound cYY in wild-type CYP121A1, as well as mutants of the active site aromatic residues F168 and W182 that have previously been characterized in vitro. Of note, simulated changes in cYY orientation align closely with changes in CYP121A1 catalysis described in vitro. For example, the mutant W182Y allows cYY to achieve proximity to the heme, which we posit models a catalytically relevant binding mode. Interestingly, a similar binding mode was observed for a single CYP121 protomer in simulations of the intact dimer. These findings, including in vitro analysis of the active site mutation R386N, inform a model of the multistep dynamics of bound cYY and address multiple unresolved questions raised in prior studies while also informing the development of future drug design by highlighting additional orientations of bound cYY that correlate with CYP121A1 function.