Polyphenols in Inner Ear Neurobiology, Health and Disease: From Bench to Clinics.

There is substantial experimental and clinical interest in providing effective ways to both prevent and slow the onset of hearing loss. Auditory hair cells, which occur along the basilar membrane of the cochlea, often lose functionality due to age-related biological alterations, as well as from exposure to high decibel sounds affecting a diminished/damaged auditory sensitivity. Hearing loss is also seen to take place due to neuronal degeneration before or following hair cell destruction/loss. A strategy is necessary to protect hair cells and XIII cranial/auditory nerve cells prior to injury and throughout aging. Within this context, it was proposed that cochlea neural stem cells may be protected from such aging and environmental/noise insults via the ingestion of protective dietary supplements. Of particular importance is that these studies typically display a hormetic-like biphasic dose-response pattern that prevents the occurrence of auditory cell damage induced by various model chemical toxins, such as cisplatin. Likewise, the hormetic dose-response also enhances the occurrence of cochlear neural cell viability, proliferation, and differentiation. These findings are particularly important since they confirmed a strong dose dependency of the significant beneficial effects (which is biphasic), whilst having a low-dose beneficial response, whereas extensive exposures may become ineffective and/or potentially harmful. According to hormesis, phytochemicals including polyphenols exhibit biphasic dose-response effects activating low-dose antioxidant signaling pathways, resulting in the upregulation of vitagenes, a group of genes involved in preserving cellular homeostasis during stressful conditions. Modulation of the vitagene network through polyphenols increases cellular resilience mechanisms, thus impacting neurological disorder pathophysiology. Here, we aimed to explore polyphenols targeting the NF-E2-related factor 2 (Nrf2) pathway to neuroprotective and therapeutic strategies that can potentially reduce oxidative stress and inflammation, thus preventing auditory hair cell and XIII cranial/auditory nerve cell degeneration. Furthermore, we explored techniques to enhance their bioavailability and efficacy.

Mammalian Flavoproteome Analysis Using Label-Free Quantitative Mass Spectrometry.

Human flavin cofactor-containing enzymes constitute a small, but highly important flavoproteome. Its stability is required to ensure key metabolic functions, such as oxidative phosphorylation and beta-oxidation of fatty acid. Flavoproteome disfunction due to mutations of individual proteins or because of the lack of FMN and FAD precursor riboflavin (vitamin B2) results in clinically relevant abnormal cellular states and diseases. Current technical possibilities in the field of the quantitative mass spectrometry of proteins allow studying the flavoproteome changes under different stress conditions, including the deficiency of vitamin B2. The biological readouts of flavoenzyme destabilization, such as protein degradation and aggregation, provide important insights into the molecular mechanisms of metabolic adaptation to nutrient deficiency. The proteomic-scale studies of protein stability have significant novelty potential in basic and applied biomedical research.

Ferroptosis-Related Flavoproteins: Their Function and Stability.

Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of diverse diseases. From the other side, the induction of ferroptosis can be used to kill tumor cells as a novel therapeutic approach. Because of the broad clinical relevance, a comprehensive understanding of the ferroptosis-controlling protein network is necessary. Noteworthy, several proteins from this network are flavoenzymes. This review is an attempt to present the ferroptosis-related flavoproteins in light of their involvement in anti-ferroptotic and pro-ferroptotic roles. When available, the data on the structural stability of mutants and cofactor-free apoenzymes are discussed. The stability of the flavoproteins could be an important component of the cellular death processes.

The protective role of m1A during stress-induced granulation.

Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

Flavin dependency undermines proteome stability, lipid metabolism and cellular proliferation during vitamin B2 deficiency.

Tumor cells adapt their metabolism to meet the energetic and anabolic requirements of high proliferation and invasiveness. The metabolic addiction has motivated the development of therapies directed at individual biochemical nodes. However, currently there are few possibilities to target multiple enzymes in tumors simultaneously. Flavin-containing enzymes, ca. 100 proteins in humans, execute key biotransformations in mammalian cells. To expose metabolic addiction, we inactivated a substantial fraction of the flavoproteome in melanoma cells by restricting the supply of the FMN and FAD precursor riboflavin, the vitamin B2. Vitamin B2 deficiency affected stability of many polypeptides and thus resembled the chaperone HSP90 inhibition, the paradigmatic multiple-target approach. In support of this analogy, flavin-depleted proteins increasingly associated with a number of proteostasis network components, as identified by the mass spectrometry analysis of the FAD-free NQO1 aggregates. Proteome-wide analysis of the riboflavin-starved cells revealed a profound inactivation of the mevalonate pathway of cholesterol synthesis, which underlines the manifold cellular vulnerability created by the flavoproteome inactivation. Cell cycle-arrested tumor cells became highly sensitive to alkylating chemotherapy. Our data suggest that the flavoproteome is well suited to design synthetic lethality protocols combining proteostasis manipulation and metabolic reprogramming.

Polylysine is a Proteostasis Network-Engaging Structural Determinant.

C-terminal polylysine (PL) can be synthesized from the polyadenine tail of prematurely cleaved mRNAs or when a read-though of a stop codon happens. Due to the highly positive charge, PL stalls in the electrostatically negative ribosomal exit channel. The stalled polypeptide recruits the Ribosome-associated quality control (RQC) complex which processes and extracts the nascent chain. Dysfunction of the RQC leads to the accumulation of PL-tagged proteins, induction of a stress response, and cellular toxicity. Not much is known about the PL-specific aspect of protein quality control. Using quantitative mass spectrometry, we uncovered the post-ribosomal PL-processing machinery in human cytosol. It encompasses key cytosolic complexes of the proteostasis network, such as chaperonin TCP-1 ring complexes (TRiC) and half-capped 19S-20S proteasomes. Furthermore, we found that the nuclear transport machinery associates with PL, which suggests a novel mechanism by which faulty proteins can be compartmentalized in the cell. The enhanced nuclear import of a PL-tagged polypeptide confirmed this implication, which leads to questions regarding the biological rationale behind it.

Recognition of enzymes lacking bound cofactor by protein quality control.

Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor binding-related enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders.

Quantitative proteomics reveals that Hsp90 inhibition preferentially targets kinases and the DNA damage response.

Despite the increasing importance of heat shock protein 90 (Hsp90) inhibitors as chemotherapeutic agents in diseases such as cancer, their global effects on the proteome remain largely unknown. Here we use high resolution, quantitative mass spectrometry to map protein expression changes associated with the application of the Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). In depth data obtained from five replicate SILAC experiments enabled accurate quantification of about 6,000 proteins in HeLa cells. As expected, we observed activation of a heat shock response with induced expression of molecular chaperones, which refold misfolded proteins, and proteases, which degrade irreparably damaged polypeptides. Despite the broad range of known Hsp90 substrates, bioinformatics analysis revealed that particular protein classes were preferentially affected. These prominently included proteins involved in the DNA damage response, as well as protein kinases and especially tyrosine kinases. We followed up on this observation with a quantitative phosphoproteomic analysis of about 4,000 sites, which revealed that Hsp90 inhibition leads to much more down- than up-regulation of the phosphoproteome (34% down versus 6% up). This study defines the cellular response to Hsp90 inhibition at the proteome level and sheds light on the mechanisms by which it can be used to target cancer cells.

Protein folding in the cytoplasm and the heat shock response.

Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.