Biological Chemistry最新文献

Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into mitochondria. If the rate of protein synthesis exceeds the capacity of the mitochondrial import machinery, precursor proteins can transiently accumulate in the cytosol. The cytosolic accumulation of mitochondrial precursors jeopardizes cellular protein homeostasis (proteostasis) and can be the cause of diseases. In order to prevent these toxic effects, most non-imported precursors are rapidly degraded by the ubiquitin-proteasome system. However, cells employ a second layer of defense which is the facilitated sequestration of mitochondrial precursor proteins in transient protein aggregates. The formation of such structures is triggered by nucleation factors such as small heat shock proteins. Disaggregases and chaperones can liberate precursors from cytosolic aggregates to pass them on to the mitochondrial import machinery or, under persistent stress conditions, to the proteasome for degradation. Owing to their role as transient buffering systems, these aggregates were referred to as MitoStores. This review articles provides a general overview about the MitoStore concept and the early stages in mitochondrial protein biogenesis in yeast and, in cases where aspects differ, in mammalian cells.

The heat stress response is an essential defense mechanism in all organisms. Heat shock proteins (Hsps) are produced in response to thermal stress, with their expression levels regulated by heat shock transcription factors. In Escherichia coli, the key transcription factor σ³² positively regulates Hsp expression. Studies from over two decades ago revealed that σ³² abundance is negatively controlled under normal conditions, mainly through degradation mechanisms involving DnaK, GroEL, and FtsH. Beyond this established mechanism, recent findings indicate that a small heat shock protein IbpA also plays a role in the translational regulation of σ³², adding a new layer to the established model. This review highlights the role of a new actor, IbpA, which strongly suppresses σ³² expression under non-stress conditions and markedly increases it during heat shock.

Ischemia/reperfusion injury (I/R) is a leading cause of acute kidney injury (AKI) in conditions like kidney transplants, cardiac surgeries, and nephrectomy, contributing to high global mortality and morbidity. This study aimed to analyze the protective effects of 2'-hydroxychalcones in treating I/R-induced AKI by targeting key pathological pathways. Considering strong antioxidant action along with other pharmacological roles of chalcone derivatives, six 2'-hydroxychalcones were synthesized via Claisen-Schmidt condensation and analyzed for their protective effects in an I/R induced AKI model using HK-2 cells. Among six 2'-hydroxychalcones, chalcone A4 significantly increased the HK-2 cells viability compared to I/R group. Chalcone A4 reduced the cell death events by reducing generation of cytoplasmic ROS and mitochondrial transmembrane potential. It also increased GSH and SOD activity while reducing TBARS levels, indicating strong antioxidant action. Scanning electron microscope images showed that chalcone A4 reversed I/R-induced morphological changes in HK-2 cells, including apoptotic blebbing and cytoplasmic fragmentation. Furthermore, in silico studies revealed interactions with NADPH oxidase 4, further supporting its protective role in I/R-induced AKI. These results showed that chalcone A4 possess potential protective action against I/R induced cellular damage possibly due to its strong antioxidant action and potential interaction with NOX4 subunit of NADPH oxidase.

Kallikrein-related peptidase 7 (KLK7) is one of 15 members of the tissue kallikrein family and is primarily expressed in the skin epidermis. The activity of KLK7 is tightly regulated by multiple stages of maturation and reversible inhibition, similar to several other extracellular proteases. In this work, we used protease-specific inhibitors and active site variants to show that KLK7 undergoes autolysis at two separate sites in the 170 and 99 loops (chymotrypsinogen numbering), resulting in a loss of enzymatic activity. A protein BLAST search using the autolyzed KLK7 loop sequences identified mast cell chymase as a potential KLK7 substrate. Indeed, KLK7 cleaves chymase resulting in a concomitant loss of activity. We further demonstrate that KLK7 can hydrolyze other mast cell proteases as well as several cytokines. These cytokines belong mainly to the interferon and IL-10 families including IFN-α, IFN-β, IFN-γ, IL-28A/IFN-λ2, IL-20, IL-22, and IL-27. This is the first study to identify a possible molecular interaction link between KLK7 and mast cell proteases and cytokines. Although the precise biological implications of these findings are unclear, this study extends our understanding of the delicate balance of proteolytic regulation of enzyme activity that maintains physiological homeostasis, and facilitates further biological investigations.

Heat stress impacts male reproduction in animal husbandry. Carnosic acid (CA), a potent antioxidant, mitigates oxidative stress and apoptosis. αB-crystallin, a small heat shock protein, regulates apoptosis and oxidative stress. This study examines the protective effects of CA on the testis in wild-type and αB-crystallin knockout mice under heat stress. CA pretreatment increased testosterone levels and preserved testicular structure in wild-type mice, but no changes in knockout mice. CA reduced Hsp27, Hsp70, and cleaved caspase-3 levels, while knockout mice showed increased cleaved caspase-3. These results suggest that CA protects the testis by modulating heat shock and apoptosis-related proteins.

The amyloid precursor protein (APP) can be modulated by the binding of copper and zinc ions. Both ions bind with low nanomolar affinities to both subdomains (E1 and E2) in the extracellular domain of APP. However, the impact of ion binding on structural and mechanical trans-dimerization properties is yet unclear. Using a bead aggregation assay (BAA), we found that zinc ions increase the dimerization of both subdomains, while copper promotes only dimerization of the E1 domain. In line with this, scanning force spectroscopy (SFS) analysis revealed an increase in APP adhesion force up to three-fold for copper and zinc. Interestingly, however, copper did not alter the separation length of APP dimers, whereas high zinc concentrations caused alterations in the structural features and a decrease of separation length. Together, our data provide clear differences in copper and zinc mediated APP trans-dimerization and indicate that zinc binding might favor a less flexible APP structure. This fact is of significant interest since changes in zinc and copper ion homeostasis are observed in Alzheimer's disease (AD) and were reported to affect synaptic plasticity. Thus, modulation of APP trans-dimerization by copper and zinc could contribute to early synaptic instability in AD.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.

Sensory neurons serve to receive and transmit a wide range of information about the conditions of the world around us as well as the external and internal state of our body. Sensitisation of these nerve cells, i.e. becoming more sensitive to stimuli or the emergence or intensification of spontaneous activity, for example in the context of inflammation or nerve injury, can lead to chronic diseases such as neuropathic pain. For many of these disorders there are only very limited treatment options and in order to find and establish new therapeutic approaches, research into the exact causes of sensitisation with the elucidation of the underlying mechanisms and the identification of the molecular components is therefore essential. These components include plasma membrane receptors and ion channels that are involved in signal reception and transmission. Members of the transient receptor potential (TRP) channel family are also expressed in sensory neurons and some of them play a crucial role in temperature perception. This review article focuses on the heat-sensitive TRPM3 and the cold-sensitive TRPM8 (and TRPA1) channels and their importance in sensitisation of dorsal root ganglion sensory neurons is discussed based on studies related to inflammation and injury- as well as chemotherapy-induced neuropathy.

Cytochrome P450 monooxygenases are recognized as versatile biocatalysts due to their broad reaction capabilities. One important reaction is the hydroxylation of non-activated C-H bonds. The subfamily CYP153A is known for terminal hydroxylation reactions, giving access to functionalized aliphatics. Whilst fatty derivatives may be converted by numerous enzyme classes, midchain aliphatics are seldomly accepted, a prime property of CYP153As. We report here on a new CYP153A member from the genome of the mesophilic actinobacterium Gordonia rubripertincta CWB2 as an efficient biocatalyst. The gene was overexpressed in Escherichia coli and fused with a surrogate electron transport system from Acinetobacter sp. OC4. This chimeric self-sufficient whole-cell system could perform hydroxylation and epoxidation reactions: conversions of C6-C14 alkanes, alkenes, alcohols and of cyclic compounds were observed, yielding production rates of, e.g., 2.69 mM h^-1 for 1-hexanol and 4.97 mM h^-1 for 1,2-epoxyhexane. Optimizing the linker compositions between the protein units led to significantly altered activity. Balancing linker length and flexibility with glycine-rich and helix-forming linker units increased 1-hexanol production activity to 350 % compared to the initial linker setup with entirely helical linkers. The study shows that strategic coupling of efficient electron supply and a selective enzyme enables previously challenging monooxygenation reactions of midchain aliphatics.

Posttranslational modifications (PTMs) can modulate the activity, localization and interactions of proteins and (re)define their biological function. Understanding how changing environments can alter cellular processes thus requires detailed knowledge about the dynamics of PTMs in time and space. A PTM that gained increasing attention in the last decades is protein persulfidation, where a cysteine thiol (-SH) is covalently bound to sulfane sulfur to form a persulfide (-SSH). The precise cellular mechanisms underlying the presumed persulfide signaling in plants are, however, only beginning to emerge. In the mitochondrial matrix, strict regulation of persulfidation and H₂S homeostasis is of prime importance for maintaining mitochondrial bioenergetic processes because H₂S is a highly potent poison for cytochrome c oxidase. This review summarizes the current knowledge about protein persulfidation and corresponding processes in mitochondria of the model plant Arabidopsis. These processes will be compared to the respective processes in non-plant models to underpin similarities or highlight apparent differences. We provide an overview of mitochondrial pathways that contribute to H₂S and protein persulfide generation and mechanisms for H₂S fixation and de-persulfidation. Based on current proteomic data, we compile a plant mitochondrial persulfidome and discuss how persulfidation may regulate protein function.