Biological Chemistry最新文献

Long non-coding RNAs have gained attention in recent years as they were shown to play crucial roles in the regulation of cellular processes, but the understanding of the exact mechanisms is still incomplete in most cases. This is also true for long non-coding RNA LINC00941, which was recently found to be highly upregulated in various types of cancer influencing cell proliferation and metastasis. Initial studies could not elucidate the mode of action to understand the role and real impact of LINC00941 in tissue homeostasis and cancer development. However, recent analyses have demonstrated multiple potential modes of action of LINC00941 influencing the functionality of various cancer cell types. Correspondingly, LINC00941 was proposed to be involved in regulation of mRNA transcription and modulation of protein stability, respectively. In addition, several experimental approaches suggest a function of LINC00941 as competitive endogenous RNA, thus acting in a post-transcriptional regulatory fashion. This review summarizes our recent knowledge about the mechanisms of action of LINC00941 elucidated so far and discusses its putative role in miRNA sequestering processes. In addition, the functional role of LINC00941 in regulating human keratinocytes is discussed to also highlight its role in normal tissue homeostasis tissue aside from its involvement in cancer.

Mitochondria are the essential players in eukaryotic ATP production by oxidative phosphorylation, which relies on the maintenance and accurate expression of the mitochondrial genome. Even though the basic principles of translation are conserved due to the descendance from a bacterial ancestor, some deviations regarding translation factors as well as mRNA characteristics and the applied genetic code are present in human mitochondria. Together, these features are certain challenges during translation the mitochondrion has to handle. Here, we discuss the current knowledge regarding mitochondrial translation focusing on the termination process and the associated quality control mechanisms. We describe how mtRF1a resembles bacterial RF1 mechanistically and summarize in vitro and recent in vivo data leading to the conclusion of mtRF1a being the major mitochondrial release factor. On the other hand, we discuss the ongoing debate about the function of the second codon-dependent mitochondrial release factor mtRF1 regarding its role as a specialized termination factor. Finally, we link defects in mitochondrial translation termination to the activation of mitochondrial rescue mechanisms highlighting the importance of ribosome-associated quality control for sufficient respiratory function and therefore for human health.

Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein-protein and protein-lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.

Na⁺/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Neuromuscular cell culture models are used to investigate synapse formation and function, as well as mechanisms of de-and regeneration in neuromuscular diseases. Recent developments including 3D culture technique and hiPSC technology have propelled their ability to complement insights from in vivo models. However, most cultures have not considered Schwann cells, the glial part of NMJs. In the following, a brief overview of different types of neuromuscular cocultures is provided alongside examples for studies that included Schwann cells. From these, findings concerning the effects of Schwann cells on those cultures are summarized and future lines of research are proposed.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na⁺-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k ₁ = 0.49 ± 0.28 s^-1 and k ₂ = 0.014 ± 0.003 s^-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k ₂ < 2 × 10^-4 s^-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.