Biochemistry [JOURNAL]
Sun
200 papers
Enzyme Kinetic Analysis for the 21st Century.
Marko I, Johnson KA
Biochemistry · 2026 Mar · PMID 41788058 · Publisher ↗
For much of the 20th century, enzyme kinetic analysis relied on deriving simplified rate equations under the steady-state approximation and later by analytical integration of differential equations for transient kinetics...
For much of the 20th century, enzyme kinetic analysis relied on deriving simplified rate equations under the steady-state approximation and later by analytical integration of differential equations for transient kinetics. This approach has since been surpassed by computational methods using numerical integration of rate equations to directly fit experimental data based on a complete user-defined model. This paradigm shift removes the constraints imposed by solving analytical equations, enabling far greater flexibility in experimental design and model complexity. Modern global fitting methods allow data from diverse experiments to be analyzed simultaneously using the minimum number of parameters supported by the information content of the data set. Global data fitting is more than just an algorithm for data analysis─it represents a fundamental change in how we design and interpret experiments, and eliminates many of the restrictions, approximations, and ambiguities inherent to equation-based analyses. In this review, we describe the principles and practice of global data fitting, compare the outcomes to conventional equation-based methods, and demonstrate its power through examples involving multiple experiments with distinct conditions and readouts. We explain why the common practice of making measurements in triplicate introduces uncertainty and we outline advanced methods for rigorously estimating errors in measurement and for establishing robust confidence limits on fitted parameters.
The Power of Protein Dynamics in Binding and Allostery.
Lee AL, Sapienza PJ
Biochemistry · 2026 Mar · PMID 41780056 · Full text
Protein dynamics─the fluctuating nature of protein structure once described by Gregorio Weber as "kicking and screaming"─is understood to be an intrinsic feature of proteins and their function. Yet it is often difficult...
Protein dynamics─the fluctuating nature of protein structure once described by Gregorio Weber as "kicking and screaming"─is understood to be an intrinsic feature of proteins and their function. Yet it is often difficult to pin down exactly how those dynamics assist function. Allosteric regulation is a widespread protein function that was once seen to operate solely through conformational change. Over the last two decades, a series of experimental studies has shown that thermally activated, rapid-time scale dynamics can underlie allosteric ligand binding cooperativity, even in the absence of conformational change. This concept is known as "dynamic allostery", in which localized dynamics represent conformational entropy that can effectively serve as a set of thermodynamic "nano-levers". Here, we review these studies and their collective finding: that changes in the amplitudes of picosecond-nanosecond timescale side-chain dynamics can exert a large entropic driving force in protein binding events. The studies require NMR relaxation measurements of methyl "order parameters" (). We focus on the recent example from Sgt2, a chaperone in yeast's guided tail-anchoring protein pathway. Sgt2 harbors an intrinsically disordered C-terminal tail that allosterically enhances side-chain dynamics in other domains, which in turn abrogates binding to partner Get4/5. Motivated by this example, order parameters are explained in simple terms and discussed empirically to raise confidence in them as meaningful reporters of local motion. Specific studies are highlighted to show that different proteins utilize distinct dynamic strategies for allosteric coupling. Finally, the surprising role of disordered tails in controlling dynamic allostery is discussed.
The Reductive Power of Flavin Mononucleotide Does Not Dictate the Product Profile of the Nitroreductase, NfsA.
Cheema EK, Zou D, Rokita SE
Biochemistry · 2026 Mar · PMID 41779958 · Publisher ↗
Flavin mononucleotide (FMN)-dependent nitroreductases offer a mild and selective route to reduce nitroaromatic compounds, yet these typically fail to generate the corresponding amines. This incomplete transformation has...
Flavin mononucleotide (FMN)-dependent nitroreductases offer a mild and selective route to reduce nitroaromatic compounds, yet these typically fail to generate the corresponding amines. This incomplete transformation has been attributed to the two-electron redox potential of FMN. To test this hypothesis, the major nitroreductase NfsA from was reconstituted with a series of FMN variants spanning midpoint potentials of -215 to -307 mV. Product profiles were examined with four substrates covering a range of electron affinities (nitrofurazone, 1,3-dinitrobenzene, 4-nitroacetophenone, and nitrobenzene), and all combinations of enzymes and substrates were found to yield only the hydroxylamine products. No amines were detected under any condition, and as a confirmation, 4-hydroxylaminacetophenone was shown to be inert to treatment with reduced nicotinamide adenine dinucleotide phosphate and NfsA reconstituted with a low-potential FMN variant (-295 mV). The inability of NsfA to generate amines is consequently not a function of the reducing potential. However, this is a determinant of the catalytic efficiency. The for nitroacetophenone turnover decreased almost 240-fold for NfsA containing an FMN variant with an of -307 mV relative to that containing native FMN (-215 mV). 1,3-Dinitrobenzene experienced the smallest decrease of 52-fold in the same comparison. Redox tuning of NfsA can therefore be detrimental to catalytic efficiency and fails to generate the desired amine products. A renewed focus on active site properties is recommended for engineering new catalysts to promote nitroaromatic to arylamine conversion.
Unravelling the Enantioselective Mechanism of Benzylsuccinate Synthase: Insights into Anaerobic Hydrocarbon Degradation through Multiscale Modeling and Microkinetics.
Szaleniec M, Oleksy G, Borowski T … +1 more , Heider J
Biochemistry · 2026 Mar · PMID 41775511 · Full text
Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of -benzylsuccinat...
Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of -benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present a detailed theoretical study of the reaction mechanism using classical molecular dynamics and multiscale modeling (QM/MM). We describe the potential energy surface of the reaction and confirm the previously postulated mechanism. However, the multiscale character of our model allowed us to elucidate the origins of several experimentally observed catalytic phenomena, such as the inversion of the benzylic carbon configuration upon C-C bond formation and the addition of the abstracted H atom back to the benzylsuccinyl radical. The obtained model is supported by microkinetic analysis and was able to explain and quantitatively predict the strict -enantioselectivity of BSS, which is enforced predominantly by the dynamic kinetic behavior of toluene in the active site, leading to over 40-times faster production of the -enantiomer, not by the binding orientation of the fumarate. Our study contributes to the elucidation of the catalytic processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.
Manipulating the Unfolded State of a Folded Protein through Site-Specific Backbone Modification.
Page GE, Lin Y, Horne WS
Biochemistry · 2026 Mar · PMID 41773780 · Full text
Protein unfolded states are heterogeneous but can manifest local and long-range order. Replacement of side chains through site-directed mutagenesis is a common method to manipulate the unfolded state and elucidate its ro...
Protein unfolded states are heterogeneous but can manifest local and long-range order. Replacement of side chains through site-directed mutagenesis is a common method to manipulate the unfolded state and elucidate its role in the folding process. Modification of the protein backbone represents a less explored complementary approach with the potential to elicit dramatic changes in conformational preferences from minimal chemical alteration. Prior work has shown backbone modification can affect unfolded ensembles as well as intrinsically disordered sequences. Here, we show that it can be used to rationally tune structural characteristics of the unfolded state of a folded protein. Using the GCN4 leucine zipper as a host, canonical α-residues throughout the chain are individually replaced by β or C-Me-α analogues. The former modification enhances conformational freedom, the latter restricts it, and both retain the side chain at the substitution site. Characterization by circular dichroism and X-ray crystallography shows that the variants adopt folded structures identical to the prototype. Thermal and thermodynamic stability vary in complex ways with the context and nature of backbone modification; however, a uniform relationship is observed between substitution type and the sensitivity of folding free energy to chemical denaturant. This finding suggests systematic changes in solvent-accessible surface area of the unfolded ensemble among isomeric proteins differing only in the position of a single CH group. Collectively, these results demonstrate a platform for predictably tuning the properties of the unfolded state through minimal chemical modification, enabling new avenues for fundamental research on folding behavior of proteins as well as protein mimetics.
Dynamic Hotspots in the Uba7 Ubiquitin-Fold Domain Direct UbcH8 Recognition.
Dağ Ç, Lambert M, Kazar AE … +10 more , Kahraman K, Göcenler O, Lee W, Tozkoparan Ceylan CD, Löhr F, Shim JG, Haas AL, Dötsch V, Ziarek J, Elgin ES
Biochemistry · 2026 Mar · PMID 41773046 · Publisher ↗
ISGylation is a ubiquitin-like post-translational modification that plays a central role in innate immune signaling. Conjugation of interferon-stimulated gene 15 (ISG15) to target proteins is initiated by the E1 enzyme U...
ISGylation is a ubiquitin-like post-translational modification that plays a central role in innate immune signaling. Conjugation of interferon-stimulated gene 15 (ISG15) to target proteins is initiated by the E1 enzyme Uba7, transferred to the E2 enzyme UbcH8, and completed by an E3 ligase. Specificity in this cascade is mediated by the ubiquitin-fold domain (UFD) of Uba7, yet the structural and mechanistic basis of E1-E2 recognition remains poorly defined. Here, we present the solution NMR structure and functional characterization of a human Uba7-UFD. NMR chemical shift perturbation experiments combined with site-directed mutagenesis delineate the UbcH8 interaction surface and identify residues critical for E1-E2 binding. The Uba7-UFD adopts a conserved ubiquitin-fold architecture but exhibits conformational flexibility in the unbound state. N relaxation measurements show a globally well-folded domain with localized ps-ns time scale dynamics within the β2/β4 E2 binding surface and the acidic loop spanning residues 996-1008. Upon UbcH8 binding, relaxation parameters shift toward those expected for a larger effective molecular size, accompanied by an increased residue-specific heterogeneity at the interface, consistent with binding-coupled changes in local mobility. Mutational analysis identifies C996 as being essential for UFD structural integrity and binding competence. Moreover, targeted alterations in the length and flexibility of the adjacent acidic loop strongly impair UbcH8 binding, demonstrating that the loop architecture is a critical determinant of efficient E2 recruitment. Together, these results provide a structural and dynamic framework for understanding E2 enzyme selection in the ISGylation pathway and highlight the role of UFD conformational dynamics in the E1-E2 complex formation.
Cargo Recognition of Nesprin-2 by the Dynein Adapter Bicaudal D2 for a Nuclear Positioning Pathway That Is Important for Brain Development.
Rodriguez Castro ED, Putta S, Ali MY … +5 more , Garcia Martin JM, Zhao X, Sylvain S, Trybus KM, Solmaz SR
Biochemistry · 2026 Mar · PMID 41770881 · Full text
Nesprin-2 and its paralog Nesprin-1 are subunits of LINC complexes that are essential for brain development. To position the nucleus for neuronal migration, Nesprin-2 interacts with the motors kinesin-1 and dynein, which...
Nesprin-2 and its paralog Nesprin-1 are subunits of LINC complexes that are essential for brain development. To position the nucleus for neuronal migration, Nesprin-2 interacts with the motors kinesin-1 and dynein, which are recruited by the adapter Bicaudal D2 (BicD2), but the molecular details of these interactions are elusive. Here, structural models of minimal Nesprin-2/BicD2 complexes with 1:2 and 2:2 stoichiometry were predicted using AlphaFold and experimentally validated by mutagenesis, binding assays, and single-molecule biophysical studies. The core of the binding site is formed by spectrin repeats of Nesprin-2, which form an α-helical bundle with BicD2 that is structurally distinct from the Rab6/BicD2 and Nup358/BicD2 complexes. Such structural differences could fine-tune the motility of associated dynein and kinesin-1 motors for these transport pathways. Furthermore, the Nesprin-2 fragment interacts with full-length BicD2 and activates dynein/dynactin/BicD2 complexes for processive motility, suggesting that no additional components are required to reconstitute this transport pathway. Interestingly, either one or two Nesprin-2 molecules can bind to a BicD2 dimer and activate BicD2/dynein/dynactin complexes for processive motion, resulting in similar speed and run lengths. The BicD2/dynein binding site is spatially close but does not overlap with the kinesin-1 recruitment site, thus both motors may interact with Nesprin-2 simultaneously. Several mutations of Nesprin-1 and 2 that cause Emery-Dreifuss muscular dystrophy are found in the motor-recruiting domain and may alter interactions with kinesin-1 and BicD2/dynein, consistent with the abnormally positioned nuclei found in patients with this disease.
Dissecting the Binding Interactions of the Chromatin Remodeler SMARCA4 with G-Quadruplex DNA.
Madden SK, Tannahill D, Balasubramanian S
Biochemistry · 2026 Mar · PMID 41759551 · Full text
DNA G-quadruplexes (G4s) are key structural features in chromatin that are important to genome function. G4s have an apparent capacity to recruit a wide variety of proteins, including chromatin remodelers, yet the molecu...
DNA G-quadruplexes (G4s) are key structural features in chromatin that are important to genome function. G4s have an apparent capacity to recruit a wide variety of proteins, including chromatin remodelers, yet the molecular basis and biophysical principles governing these interactions remain poorly understood. Here, we sought to build insights into the interactions of chromatin remodeler SMARCA4 with G4s using a biophysical approach. We found that SMARCA4 selectively recognizes the G4 structure over duplex and single-stranded DNA. SMARCA4 binds a wide range of G4s with different topologies and loop lengths with similar low nanomolar affinities. SMARCA4 was also observed to have a longer residency time on the G4 structure compared to that of other known protein-DNA interactions. We also found that the D1 (DExx-c) helicase domain of SMARCA4, which is important for tethering SMARCA4 to chromatinized DNA, was the predominant binding domain for G4 recognition. Our findings reveal new insights into how G4s interact with proteins, which may have important implications for understanding G4-mediated genome mechanisms.
5,10-Methylenetetrahydrofolate Reductase─the Key Allosteric Regulator in One-Carbon Metabolism.
Blomgren LKM, Guo S, Froese DS … +2 more , McCorvie TJ, Yue WW
Biochemistry · 2026 Mar · PMID 41758688 · Full text
Collectively known as one-carbon metabolism (OCM), both the folate and methionine cycles are highly regulated to meet cellular demands. These cycles are key in the production and recycling of methyl groups to be used in...
Collectively known as one-carbon metabolism (OCM), both the folate and methionine cycles are highly regulated to meet cellular demands. These cycles are key in the production and recycling of methyl groups to be used in many essential cellular processes such as the production of nucleotides, as well as -adenosyl-l-methionine (SAM) the global methyl donor for DNA, RNA, and post translational modifications. Within the folate cycle, 5,10-methylenetetrahydrofolate is the main species through which methyl groups enter OCM. Therefore, 5,10-methylenetetrahydrofolate reductase (MTHFR), which reduces 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, is the central enzyme that directs methyl groups for use within the methionine cycle. MTHFR is an enzyme found in all domains of life, but unlike in prokaryotes, eukaryotic MTHFR activity is highly regulated by the level of SAM, to balance the one-carbon needs of the cell. In this perspective, we review the catalytic mechanism of MTHFR, evolutionary differences, and the regulatory mechanisms that have evolved to alter its activity. We also discuss recent structural findings that reveal a unique mechanism for inactivation by SAM as a feedback loop and its consequences for understanding inherited MTHFR deficiency.
Characterization of Conformational Dynamics and Structural Plasticity of the Catalytic Domain of Human Mitochondrial YME1L Protease.
Black MK, Kim A, Chen CY … +4 more , Goncalves MM, Waseem S, Vahidi S, Huang R
Biochemistry · 2026 Mar · PMID 41746830 · Publisher ↗
Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L is a membrane-anchored AAA+ (TPases ssociated with diverse cellular ctivities) family protease and plays a pivotal role in mitocho...
Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L is a membrane-anchored AAA+ (TPases ssociated with diverse cellular ctivities) family protease and plays a pivotal role in mitochondrial proteostasis by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1L's conformational dynamics, proteolytic activity, and stability remain unclear. Here, we characterize the conformational dynamics of the YME1L catalytic domain. Using a hexameric soluble YME1L construct, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy to demonstrate that nucleotide binding reduces the backbone flexibility and modulates the side-chain dynamics of the AAA+ domain, while Zn binding stabilizes the protease domain. We also reveal long-range functional crosstalk between the AAA+ and protease domains of YME1L. We use functional assays to show the importance of a salt bridge between the AAA+ and protease domains in facilitating ATP-dependent substrate degradation by YME1L. Additionally, we show that ATP binding stabilizes the structure of the catalytic domain of YME1L and protects it from chemical- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide insights into our understanding of its proteolytic activity and structural stability under stress conditions.
Corrections to "Discovery of the Cytocapsular Membrane as Hallmark of Malignant Tumors".
Yi T, Wagner G
Biochemistry · 2026 Mar · PMID 41742620 · Publisher ↗
Abstract loading — click title to view on PubMed.
Allostery between Distant Structural Regions Dictates Selectivity in GPCR:G Protein Coupling.
Mukhaleva E, Sánchez Rivas EJ, Branciamore S … +3 more , Rodin AS, Sivaramakrishnan S, Vaidehi N
Biochemistry · 2026 Mar · PMID 41738444 · Full text
Despite extensive structural and functional studies, the molecular mechanisms governing G-protein coupled receptor-G (GPCR-G) protein coupling selectivity remain unresolved. Here, using an interpretable machine learning...
Despite extensive structural and functional studies, the molecular mechanisms governing G-protein coupled receptor-G (GPCR-G) protein coupling selectivity remain unresolved. Here, using an interpretable machine learning Bayesian Network model with Molecular Dynamics simulations and experiments, we reveal the influence of distant residue communities within the Gα protein core on coupling selectivity. We observed distinct cooperative hotspot residues across different Gα protein subtypes, including key regions such as the N-terminus, h4s6 loop, and H5 helix. These results demonstrate the intricate allosteric dependencies between the core and the H5 helix in stabilizing selective interactions. The functional significance of these cooperative regions is validated through subtype-swapping mutations. By introducing targeted Gαq-like mutations in the Gαs core, we successfully altered the receptor coupling profile to signal through Gαq. Our findings emphasize that cooperative interactions in the Gα core are not only crucial for selectivity but can also be leveraged to engineer Gα proteins with tailored coupling preferences.
Lysyl Oxidase LOXL2 Selectively Oxidizes Primary, α-Unbranched Amines and Prefers Cationic Substrates.
Poller LM, Wennemers H
Biochemistry · 2026 Mar · PMID 41729086 · Publisher ↗
Lysyl oxidases (LOXs) initiate the posttranslational cross-linking of collagen by oxidizing lysine to allysine residues, a process crucial for the mechanical properties of the extracellular matrix. However, dysregulated...
Lysyl oxidases (LOXs) initiate the posttranslational cross-linking of collagen by oxidizing lysine to allysine residues, a process crucial for the mechanical properties of the extracellular matrix. However, dysregulated LOX levels─particularly those of the LOXL2 isoform─have been implicated in numerous fibrotic diseases and cancers. Accordingly, considerable effort has been devoted to understanding the biological role of LOXL2. Despite this interest, little is known about how the type, structure, and neighboring groups of the amine influence LOXL2 activity. Here, we determined Michaelis-Menten kinetics for a panel of lysine-based model substrates to assess the structural determinants of LOXL2-catalyzed oxidation. We show that LOXL2 oxidizes exclusively primary, α-unbranched amines. In addition, our studies revealed that an additional positively charged group enhances LOXL2 activity.
Role of Aspartate 86 in the Catalytic Mechanism of Glutamate Decarboxylase.
Giovannercole F, Pennacchietti E, Grassini G … +1 more , De Biase D
Biochemistry · 2026 Mar · PMID 41717872 · Full text
In bacteria, the pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamate decarboxylase (Gad) protects the cells exposed to an acidic environment by consuming one proton/catalytic cycle during the conversion of l-glutamat...
In bacteria, the pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamate decarboxylase (Gad) protects the cells exposed to an acidic environment by consuming one proton/catalytic cycle during the conversion of l-glutamate to γ-aminobutyrate (GABA) and CO. The enzyme (GadB) is the best-characterized bacterial Gad; its activity is maximal at pH 4-5 and undetectable at pH ≥ 6.0, at which the active site is closed by His465. The imidazole ring of this His residue, highly conserved in bacterial Gad, becomes deprotonated as the pH increases above 5.0 and carries out a nucleophilic attack on the PLP-Lys276 Schiff base. However, when His465 is mutated, GadB activity still displays pH dependence, indicating that other residues also play a role. Herein, through a combination of spectroscopic and kinetic analyses, including solvent kinetic isotope effect (SKIE) and proton inventory studies, Asp86, another residue highly conserved in bacterial Gad, was shown to play an important role in substrate binding and product release and, unexpectedly, to be a major player in the large SKIE observed in GadB. This was demonstrated by incorporating the D86N substitution into the GadB_H465A variant to avoid the masking effect of His465 at pH > 5.5. In addition, GadB_D86N-H465A was shown to be less sensitive than GadB_H465A to the pH increase occurring during the decarboxylation, being still active in the pH range 7-8, where glutamate solubility increases. This finding, together with the enzyme's improved ability to release the product, makes GadB_D86N-H465A interesting also for effective biobased synthesis of GABA.
Catalytic p Attenuation in a Hydrolytic Metalloenzyme by Genetic Code Expansion.
Manser BP, Deliz Liang A
Biochemistry · 2026 Mar · PMID 41705832 · Full text
Hydrolytic metalloenzymes employ Lewis-acidic metal cofactors to activate water molecules, generating nucleophilic hydroxide species that facilitate catalysis. Their catalytic efficiency across a wide pH range is often g...
Hydrolytic metalloenzymes employ Lewis-acidic metal cofactors to activate water molecules, generating nucleophilic hydroxide species that facilitate catalysis. Their catalytic efficiency across a wide pH range is often governed by the protonation state of the metal-bound water, reflected in p values typically between 6.8 and 9. Modulating this parameter is key to expanding enzymatic activity for improved activity at neutral to acidic pH. Herein, we apply genetic code expansion to mutate the primary metal-coordination sphere of a model metallohydrolase: the dizinc phosphotriesterase from . Substitution of the most catalytically indispensable coordinating histidine residue (H55) to -methyl-l-histidine (πMH) resulted in substantial enzyme yields, efficient metal coordination for either Zn or Co, and up to 5-fold improved tolerance to acidic conditions. Detailed mechanistic analysis revealed a systematic decrease in catalytic p and attenuation of several catalytic rate constants. These results add to the growing body of evidence demonstrating the power of ncAA-based engineering for refined tuning of enzyme properties.
Rational Design of Plant-Derived Protein Ligases with Altered Substrate Specificity.
Zhou Y, de Veer SJ, Tyler TJ … +3 more , Durek T, Rehm FBH, Craik DJ
Biochemistry · 2026 Mar · PMID 41705341 · Publisher ↗
Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced th...
Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced the transpeptidation efficiency of several enzymes, attempts to modify their substrate specificity have been more limited. In a recent study, we produced the first asparaginyl ligase with engineered P2' substrate specificity by mutating Tyr188 to Ala in AEP1. Here, we report the engineering of two additional asparaginyl ligases from different plant families, VyPAL2 and butelase 1. We show that mutating the corresponding Tyr residue located in the S2' pocket of these enzymes also expands their substrate scope, enabling the mutant enzymes to process substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling that their parent enzymes process poorly. These findings further establish the role of the conserved S2' Tyr residue as a general determinant of substrate specificity for asparaginyl ligases and provide a path toward more extensive engineering efforts.
C-Terminal Fragment of Filamin C Containing Immunoglobulin-Like Domains 19-24 Selectively Interacts with the Small Heat Shock Protein HspB7.
Zamotina MA, Muranova LK, Tyurin-Kuzmin PA … +2 more , Sluchanko NN, Gusev NB
Biochemistry (Mosc) · 2026 Jan · PMID 41702738 · Publisher ↗
Filamin C is an adapter protein involved in the regulation of cytoskeleton; it interacts with more than 90 protein partners, including small heat shock proteins (sHsps). However, the details of filamin C interaction with...
Filamin C is an adapter protein involved in the regulation of cytoskeleton; it interacts with more than 90 protein partners, including small heat shock proteins (sHsps). However, the details of filamin C interaction with sHsps remain poorly characterized. Here, we used immunochemistry methods, size-exclusion chromatography, native gel electrophoresis, and chemical crosslinking to investigate the interactions of a long C-terminal fragment of filamin C containing immunoglobulin (Ig)-like domains 19-24 (FLNC19-24) with sHsps. Out of five analyzed sHsps (HspB1, phosphorylation-mimicking 3D mutant of HspB1, HspB5, HspB6, HspB7, and HspB8), only HspB7 formed complexes with FLNC19-24. Taking into account that HspB7 interacted with the isolated Ig-like domain 24 and filamin fragments containing Ig-like domains 22-24 and 19-24, we concluded that HspB7 is a bona fide partner of filamin C. Selective binding of the α-crystallin domain of HspB7 with the Ig-like domain 24 induced dissociation of filamin dimers, which might promote filamin C translocation in the cell and facilitate the repairs of damaged contractile apparatus.
Functional Divergence of bL36m Protein in Yeast and Human Mitochondrial Ribosomes.
Piunova UE, Baleva MV, Lantsova MS … +5 more , Chicherin IV, Vasilev RA, Moysenovich AM, Levitskii SA, Kamenski PA
Biochemistry (Mosc) · 2026 Jan · PMID 41702737 · Publisher ↗
L36 is a structural protein of the large ribosomal subunit of bacterial, mitochondrial, and chloroplast ribosomes. L36 stabilizes the peptidyl transferase center and the L7/L12 stalk, which is a binding site for the elon...
L36 is a structural protein of the large ribosomal subunit of bacterial, mitochondrial, and chloroplast ribosomes. L36 stabilizes the peptidyl transferase center and the L7/L12 stalk, which is a binding site for the elongation factors during the translation cycle. According to the cryoelectron microscopy data, L36 incorporates into the large ribosomal subunit in both bacteria and mitochondria at the final assembly step. Bacterial L36 is not an essential protein, since deletion of its gene in bacteria did not impair the colony growth or reduce the mRNA translation levels. Deletion of the gene coding for the mitochondrial homologue of L36 (bL36m) in , impeded yeast growth on the media with non-fermentable carbon sources. Our findings indicate that the mitochondrial dysfunction associated with the absence of bL36m was caused by a decreased activity of cytochrome oxidase complex that resulted from the selective disruption of synthesis of its subunits encoded in the mitochondrial genome. Furthermore, selective inhibition of mitochondrial protein synthesis did not induce critical structural abnormalities of mitochondrial ribosomes or reduce their ability to bind mRNA. Furthermore, we demonstrated that in contrast to , the absence of bL36m protein in human cells had no substantial impact on the synthesis of mitochondrially encoded proteins or mitochondrial ribosome assembly. However, the observed reduction in the mitochondrial respiration in the bL36m-deficient cells may be indicative of disturbances in the respiratory chain organization not associated with the mitochondrial translation.
Regulation of Activity of DNA Polymerases β and λ by XRCC1, PARP1, and PARP2 Proteins.
Lebedeva NA, Maltseva EA, Rechkunova NI … +1 more , Lavrik OI
Biochemistry (Mosc) · 2026 Jan · PMID 41702736 · Publisher ↗
DNA damage repair by the base excision repair (BER) mechanism is a complex, multistage process that requires precise coordination and regulation of activities of enzymes at each step of DNA repair. The central stage in t...
DNA damage repair by the base excision repair (BER) mechanism is a complex, multistage process that requires precise coordination and regulation of activities of enzymes at each step of DNA repair. The central stage in this process is DNA synthesis in the gap formed by removal of the damaged nucleotide. DNA polymerases β (Pol β) and λ (Pol λ) of the X family possess all properties necessary for the DNA repair synthesis. Pol β is the major enzyme in DNA synthesis in BER, which may be due to the influence of other BER factors regulating its activity. The scaffold protein XRCC1 and poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are essential for the formation of functional BER complexes. We investigated the effect of XRCC1, PARP1, PARP2, and poly(ADP-ribosyl)ation on the activity of Pol β and Pol λ in the single-nucleotide gap filling reaction in BER. XRCC1 stimulated the activity of Pol λ to a significantly greater extent than the Pol β activity. PARP1 and PARP2 inhibited both DNA polymerases; the inhibitory effect of PARP1 was more pronounced on DNA with a tetrahydrofuran-phosphate group at the 5' end of the gap, while PARP2 effect was more significant on DNA containing a 5' phosphate group. XRCC1 partially restored the DNA polymerase activity, especially that of Pol β. It can be assumed that the XRCC1-Pol β complex competes with PARP1/2 for DNA binding more strongly than the XRCC1-Pol λ complex. Addition of NAD to PARP2 led to a more efficient restoration of Pol β (but not Pol λ) activity. These differences may be one of the causes why Pol β is the main DNA polymerase in BER.
Effect of Quizartinib on the Resistance of Acute Myeloid Leukemia Cells with FLT3-ITD-Positive and FLT3-ITD-Negative Phenotypes to the TRAIL-Induced Apoptosis.
Kobyakova MI, Lomovskaya YV, Krasnov KS … +7 more , Odinokova IV, Meshcheriakova EI, Ermakov AM, Didenko AS, Senotov AS, Fadeeva IS, Fadeev RS
Biochemistry (Mosc) · 2026 Jan · PMID 41702735 · Publisher ↗
Internal tandem duplications in the gene encoding the membrane domain of FLT3 (FLT3-ITD) are the most common genetic alteration and an unfavorable prognostic factor in the patients with acute myeloid leukemia (AML). New-...
Internal tandem duplications in the gene encoding the membrane domain of FLT3 (FLT3-ITD) are the most common genetic alteration and an unfavorable prognostic factor in the patients with acute myeloid leukemia (AML). New-generation FLT3 inhibitors effectively induce cell death in the AML cells with the FLT3-ITD-positive phenotype (FLT3-ITD) and potentially exhibit cytotoxic activity against the AML cells with the FLT3-ITD-negative phenotype (FLT3-ITD), but at higher concentrations. However, potential impact of the new-generation FLT3 inhibitors on the cytotoxic activity of molecular effectors of antitumor immunity - particularly in the context of heterogeneity of the primary clonal composition of AML, which includes both FLT3-ITD and FLT3-ITD cells - remains unclear. This study demonstrated that the use of quizartinib, a new-generation FLT3 inhibitor, increased resistance of the FLT3-ITD AML cells, but not of the FLT3-ITD AML cells, to the cytotoxic action of the key molecular effector of antitumor immunity, the cytokine Apo2L/TRAIL. This effect was mediated by the changes in the expression of proapoptotic TRAIL receptors, content of the cFLIP protein, and expression of the genes encoding proteins of the IAP and BCL-2 families. Additionally, the quizartinib-induced changes in the intracellular signaling pathways that potentially regulate TRAIL resistance in the AML cells were identified. The identified quizartinib-induced transcriptional changes are of interest not only in the context of combination therapy with TRAIL but also have broader implications for understanding the mechanisms of drug resistance in the AML cells.
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