TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Dimeric Tubulin Modifies Mechanical Properties of Lipid Bilayer, as Probed Using Gramicidin A Channel.

Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics-seen as an increase in the lifetime of the channel dimer-and thus points towards modification of the membrane's mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane's hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein-protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin.

Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Single-molecule compaction of megabase-long chromatin molecules by multivalent cations.

To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo. Variation of HO loading revealed a number of unique features related to the efficiency of chromatin compaction by multivalent cations, the mechanism of compaction, and the character of partly compact chromatin structures. The observations may be relevant for how DNA accessibility in chromatin is maintained. Compaction of saturated chromatin, in turn, is accompanied by an intra-chain segregation at the level of single chromatin molecules, suggesting an intriguing scenario of selective activation/deactivation of DNA as a result of chromatin fiber heterogeneity due to the nucleosome positioning. We suggest that this chromatin, reconstituted on megabase-long DNA because of its large size, is a useful model of eukaryotic chromatin.

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation.

The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry.

Sulforaphane is Synergistic with CB-5083 and Inhibits Colony Formation of CB-5083-Resistant HCT116 Cells.

Human p97 is a potential drug target in oncology. Mutation-driven drug resistance is an obstacle to the long-term efficacy of targeted therapy. We found that the ATPase activity for one of the CB-5083-resistant p97 mutants was reduced, which also attenuated the degradation of K48 ubiquitinated proteins in cells. To understand how p97 mutant cells with significantly reduced ATPase activity can still grow, we discovered reduced levels of CHOP and NF-κB activation in the p97 mutant cells and these cellular changes can potentially protect HCT116 cells from death due to lowered p97 activity. In addition, the NF-kB inhibitor Sulforaphane reduces proliferation of CB-5083 resistant cells and acts synergistically with CB-5083 to block proliferation of the parental HCT116 cells. The combination of Sulforaphane and CB-5083 may be a useful treatment strategy to combat CB-5083 resistance.

VDAC regulation of mitochondrial calcium flux: From channel biophysics to disease.

Voltage-dependent anion channel (VDAC), the most abundant mitochondrial outer membrane protein, is important for a variety of mitochondrial functions including metabolite exchange, calcium transport, and apoptosis. While VDAC's role in shuttling metabolites between the cytosol and mitochondria is well established, there is a growing interest in understanding the mechanisms of its regulation of mitochondrial calcium transport. Here we review the current literature on VDAC's role in calcium signaling, its biophysical properties, physiological function, and pathology focusing on its importance in cardiac diseases. We discuss the specific biophysical properties of the three VDAC isoforms in mammalian cells-VDAC 1, 2, and 3-in relationship to calcium transport and their distinct roles in cell physiology and disease. Highlighting the emerging evidence that cytosolic proteins interact with VDAC and regulate its calcium permeability, we advocate for continued investigation into the VDAC interactome at the contact sites between mitochondria and organelles and its role in mitochondrial calcium transport.

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport.

Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane, and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinson's disease (PD) related neuronal protein, alpha-synuclein (αSyn), is added to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule VDAC electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, αSyn reverses the channel's selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by αSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. -Our findings with higher calcium concentrations also demonstrate that the phenomenon of "charge inversion" is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue - VDAC1 purported calcium binding site - shows that there is no measurable effect of the mutation in either open or αSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of αSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

Targeting the Multiple Physiologic Roles of VDAC With Steroids and Hydrophobic Drugs.

There is accumulating evidence that endogenous steroids and non-polar drugs are involved in the regulation of mitochondrial physiology. Many of these hydrophobic compounds interact with the Voltage Dependent Anion Channel (VDAC). This major metabolite channel in the mitochondrial outer membrane (MOM) regulates the exchange of ions and water-soluble metabolites, such as ATP and ADP, across the MOM, thus governing mitochondrial respiration. Proteomics and biochemical approaches together with molecular dynamics simulations have identified an impressively large number of non-polar compounds, including endogenous, able to bind to VDAC. These findings have sparked speculation that both natural steroids and synthetic hydrophobic drugs regulate mitochondrial physiology by directly affecting VDAC ion channel properties and modulating its metabolite permeability. Here we evaluate recent studies investigating the effect of identified VDAC-binding natural steroids and non-polar drugs on VDAC channel functioning. We argue that while many compounds are found to bind to the VDAC protein, they do not necessarily affect its channel functions in vitro. However, they may modify other aspects of VDAC physiology such as interaction with its cytosolic partner proteins or complex formation with other mitochondrial membrane proteins, thus altering mitochondrial function.

Cells deficient for Krüppel-like factor 4 exhibit mitochondrial dysfunction and impaired mitophagy.

Krüppel-like factor 4 (Human Protein: KLF4; Human Gene: Klf4; Murine Protein: KLF4; Murine Gene: Klf4) is a zinc finger-containing transcription factor with diverse regulatory functions. Mouse embryonic fibroblasts (MEFs) lacking Klf4 exhibit genomic instability, increased reactive oxygen species (ROS), and decreased autophagy. Elevated ROS is linked to impairments in mitochondrial damage recovery responses and is often tied to disruption in mitochondrial-targeted autophagy known as mitophagy. In this study, we sought to identify a mechanistic connection between KLF4 and mitophagy. Using flow cytometry, we found that Klf4-null MEFs have diminished ability to recover mitochondrial health and regulate ROS levels after mitochondrial damage. Confocal microscopy indicated decreased localization of autophagy protein LC3 to mitochondria following mitochondrial damage in Klf4-null cells, suggesting decreased mitophagy. Western blotting and RT-PCR revealed decreased mRNA and protein expression of the mitophagy-associated protein Bnip3 and antioxidant protein GSTα4 in Klf4-null cells, providing a rationale for their impaired mitophagy and ROS accumulation. Inducing Bnip3 expression in these cells recovered mitophagy but did not decrease ROS accumulation. Our findings suggest that in Klf4-null cells, decreased Bnip3 expression impairs mitophagy and is associated with increased mitochondrial ROS production after mitochondrial damage, providing a rationale for their genomic instability and supports a tumor suppressive role for KLF4 in certain tumors as previously observed.