Exosomes are a type of extracellular vesicles, produced within multivesicular bodies, that are then released into the extracellular space through a merging of the multivesicular body with the plasma ...membrane. These vesicles are secreted by almost all cell types to aid in a vast array of cellular functions, including intercellular communication, cell differentiation and proliferation, angiogenesis, stress response, and immune signaling. This ability to contribute to several distinct processes is due to the complexity of exosomes, as they carry a multitude of signaling moieties, including proteins, lipids, cell surface receptors, enzymes, cytokines, transcription factors, and nucleic acids. The favorable biological properties of exosomes including biocompatibility, stability, low toxicity, and proficient exchange of molecular cargos make exosomes prime candidates for tissue engineering and regenerative medicine. Exploring the functions and molecular payloads of exosomes can facilitate tissue regeneration therapies and provide mechanistic insight into paracrine modulation of cellular activities. In this review, we summarize the current knowledge of exosome biogenesis, composition, and isolation methods. We also discuss emerging healing properties of exosomes and exosomal cargos, such as microRNAs, in brain injuries, cardiovascular disease, and COVID-19 amongst others. Overall, this review highlights the burgeoning roles and potential applications of exosomes in regenerative medicine.
In our recent publication, we have proposed a revised base excision repair pathway in which DNA polymerase β (Polβ) catalyzes Schiff base formation prior to the gap-filling DNA synthesis followed by ...β-elimination. In addition, the polymerase activity of Polβ employs the "three-metal ion mechanism" instead of the long-standing "two-metal ion mechanism" to catalyze phosphodiester bond formation based on the fact derived from time-resolved x-ray crystallography that a third Mg2+ was captured in the polymerase active site after the chemical reaction was initiated. In this study, we develop the models of the uncross-linked and cross-linked Polβ complexes and investigate the "three-metal ion mechanism" vs the "two-metal ion mechanism" by using the quantum mechanics/molecular mechanics molecular dynamics simulations. Our results suggest that the presence of the third Mg2+ ion stabilizes the reaction-state structures, strengthens correct nucleotide binding, and accelerates phosphodiester bond formation. The improved understanding of Polβ's catalytic mechanism provides valuable insights into DNA replication and damage repair.
To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the 2'-hydroxyl of an incoming ...ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most DNA polymerases to selectively insert deoxyribonucleotides. Recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanistic consequences of ribonucleotide misincorporation on downstream events, such as the bypass of a ribonucleotide in a DNA template and the subsequent extension of the DNA lesion bypass product. These important findings are summarized in this review.
Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer ...strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.
Proteins in vivo endure highly various interactions from the luxuriant surrounding macromolecular cosolutes. Confinement and macromolecular crowding are the two major effects that should be ...considered while comparing the results of protein dynamics from in vitro to in vivo. However, efforts have been largely focused on single domain protein folding up to now, and the quantifications of the in vivo effects in terms of confinements and crowders on modulating the structure and dynamics as well as the physical understanding of the underlying mechanisms on multidomain protein folding are still challenging. Here we developed a topology-based model to investigate folding of a multidomain Y-family DNA polymerase (DPO4) within spherical confined space and in the presence of repulsive and attractive crowders. We uncovered that the entropic component of the thermodynamic driving force led by confinements and repulsive crowders increases the stability of folded states relative to the folding intermediates and unfolded states, while the enthalpic component of the thermodynamic driving force led by attractive crowders gives rise to the opposite effects with less stability. We found that the shapes of DPO4 conformations influenced by the confinements and the crowders are quite different even when only the entropic component of the thermodynamic driving force is considered. We uncovered that under all in vivo conditions, the folding cooperativity of DPO4 decreases compared to that in bulk. We showed that the loss of folding cooperativity can promote the sequential domain-wise folding, which was widely found in cotranslational multidomain protein folding, and effectively prohibit the backtracking led by topological frustrations during multidomain protein folding processes.
Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, ...we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (G
•T/UG•T
/U
; population less than 0.4%) and one anionic species (G•T
/U
; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10
to 10
-fold discrimination against misincorporation, which proceeds primarily via tautomeric dG
•dT and dG•dT
, with contributions from anionic dG•dT
dominant at pH 8.4 and above or for some mutagenic nucleotides.
The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been ...obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in the folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to 'U-shaped' DPO4 folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4-DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, which is a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.
Liver cancer is a global health challenge as it is the third leading cause of cancer death worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is often found in ...liver cells, where it is associated with high morbidity and mortality rates. Recent studies have shown that extracellular vesicles (EVs) secreted by HCC cells play a critical role in HCC progression and metastasis. EVs contain proteins, nucleic acids, lipids, and metabolites as cargos. EVs derived from HCC cells can transfer oncogenic factors to surrounding cells leading to increased tumor growth, cell invasion, and angiogenesis. In this review, we summarize the roles that EVs play and the specific effects of their cargos on HCC progression and metastasis and identify potential therapeutic targets for HCC treatment.
During DNA polymerization, the Y-family DNA polymerases are capable of bypassing various DNA damage, which can stall the replication fork progression. It has been well acknowledged that the ...structures of the Y-family DNA polymerases have been naturally evolved to undertake this vital task. However, the mechanisms of how these proteins utilize their unique structural and conformational dynamical features to perform the translesion DNA synthesis are less understood. Here, we developed structure-based models to study the precatalytic DNA polymerization process, including DNA and nucleotide binding to DPO4, a paradigmatic Y-family polymerase from Sulfolobus solfataricus. We studied the interplay between the folding and the conformational dynamics of DPO4 and found that DPO4 undergoes first unraveling (unfolding) and then folding for accomplishing the functional “open-to-closed” conformational transition. DNA binding dynamically modulates the conformational equilibrium in DPO4 during the stepwise binding through different types of interactions, leading to different conformational distributions of DPO4 at different DNA binding stages. We observed that nucleotide binding induces modulation of a few contacts surrounding the active site of the DPO4–DNA complex associated with a high free energy barrier. Our simulation results resonate with the experimental evidence that the conformational change at the active site led by nucleotide is the rate-limiting step of nucleotide incorporation. In combination with localized frustration analyses, we underlined the importance of DPO4 conformational dynamics and fluctuations in facilitating DNA and nucleotide binding. Our findings offer mechanistic insights into the processes of DPO4 conformational dynamics associated with the substrate binding and contribute to the understanding of the “structure–dynamics–function” relationship in the Y-family DNA polymerases.
Abstract only
Emtricitabine (FTC) and lamivudine (3TC) are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in antiretroviral therapy for HIV. They contain an oxathiolane ring with ...unnatural (−)‐stereochemistry as a mimic of the ribose ring found in natural deoxynucleotides (
L
‐nucleoside). Treatment with FTC or 3TC primarily selects for the HIV‐1 reverse transcriptase (RT) M184V/I resistance mutants that are characterized by a marked decrease of incorporation of these NRTIs. Pre‐steady state kinetic data and crystal structures of both (−) and (+) oxathiolane analog triphosphates were determined in wild‐type and M184V HIV‐1 RT to elucidate the structural features of RT that recognize these substrates and gain insight into how M184V confers resistance. The pre‐steady state kinetic data reveal that (−)‐FTC‐TP and (−)‐3TC‐TP have higher binding affinities (1/
K
d
) for WT RT than dCTP however they have a slower rate of incorporation (
k
p
). The crystal structures corroborate these results revealing that the oxathiolane sulfur orients toward the DNA primer terminus and two different triphosphate orientations are observed which would reduce the rate of incorporation. The structure of (+)‐FTC‐TP shows a close match to that of the natural substrate dCTP illustrating how the oxathiolane
D
‐isomers adopt a triphosphate orientation conducive for incorporation. The M184V mutation displays significantly greater (>200‐fold)
K
d
for the
L
isomers and moderately higher (>9‐fold)
K
d
for the
D
‐isomers compared to dCTP. The structure of M184V RT illustrates how the mutation repositions the oxathiolane ring, inducing a shift of the triphosphates into a conformation that hinders nucleotide incorporation. This combined study provides a comprehensive kinetic and structural basis for inhibiting WT HIV‐1 RT by the active metabolites of 3TC and FTC and the mechanism of M184V resistance.
Support or Funding Information
This work was supported by a grant from the National Science Foundation (grant number MCB‐1716168).
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