Preterm birth prediction in asymptomatic women at mid-gestation using a panel of novel protein biomarkers: the Prediction of PreTerm Labor (PPeTaL) study.

BACKGROUND: Accurate prediction of spontaneous preterm labor/preterm birth in asymptomatic women remains an elusive clinical challenge because of the multi-etiological nature of preterm birth. OBJECTIVE: The aim of this study was to develop and validate an immunoassay-based, multi-biomarker test to predict spontaneous preterm birth. MATERIALS AND METHODS: This was an observational cohort study of women delivering from December 2017 to February 2019 at 2 maternity hospitals in Melbourne, Australia. Cervicovaginal fluid samples were collected from asymptomatic women at gestational week 16+0-24+0, and biomarker concentrations were quantified by enzyme-linked immunosorbent assay. Women were assigned to a training cohort (n = 136) and a validation cohort (n = 150) based on chronological delivery dates. RESULTS: Seven candidate biomarkers representing key pathways in utero-cervical remodeling were discovered by high-throughput bioinformatic search, and their significance in both in vivo and in vitro studies was assessed. Using a combination of the biomarkers for the first 136 women allocated to the training cohort, we developed an algorithm to stratify term birth (n = 124) and spontaneous preterm birth (n = 12) samples with a sensitivity of 100% (95% confidence interval, 76-100%) and a specificity of 74% (95% confidence interval, 66-81%). The algorithm was further validated in a subsequent cohort of 150 women (n = 139 term birth and n = 11 preterm birth), achieving a sensitivity of 91% (95% confidence interval, 62-100%) and a specificity of 78% (95% confidence interval, 70-84%). CONCLUSION: We have identified a panel of biomarkers that yield clinically useful diagnostic values when combined in a multiplex algorithm. The early identification of asymptomatic women at risk for preterm birth would allow women to be triaged to specialist clinics for further assessment and appropriate preventive treatment.

A Hexokinase 2 Modulator for Field-Directed Treatment of Experimental Actinic Keratoses.

Overexpression of hexokinase 2, and its binding to VDAC1 on the outer mitochondrial membrane of cancer cells, is key to their metabolic reprogramming to aerobic glycolysis, which enables them to proliferate. We describe Comp-1, an allosteric small molecule that selectively detaches hexokinase 2 from the mitochondria. Detachment of hexokinase 2 reduces glycolysis and triggers apoptosis in cancer cells, without affecting hexokinase 1-expressing normal cells. The anti-cancer activity of Comp-1 was demonstrated in the UVB-damaged skin model in SKH-1 mice. Topical treatment with Comp-1 led to 70% reduction in lesion number and area. This in vivo efficacy was obtained without local skin reactions or other safety findings. Mechanism-related pharmacodynamic markers, including hexokinase 2 and cleaved caspase 3 levels, are affected by Comp-1 treatment in vivo. Good Laboratory Practice toxicology studies in minipigs for 28 days and 13 weeks established no systemic toxicities and minimal dermal reaction for once-daily application of up to 20% and 15% ointment strengths, respectively. Thus, Comp-1 may address a significant unmet medical need for a non-irritating efficacious topical actinic keratosis treatment.

Novel Biomarker Proteins in Chronic Lymphocytic Leukemia: Impact on Diagnosis, Prognosis and Treatment.

In many cancers, cells undergo re-programming of metabolism, cell survival and anti-apoptotic defense strategies, with the proteins mediating this reprogramming representing potential biomarkers. Here, we searched for novel biomarker proteins in chronic lymphocytic leukemia (CLL) that can impact diagnosis, treatment and prognosis by comparing the protein expression profiles of peripheral blood mononuclear cells from CLL patients and healthy donors using specific antibodies, mass spectrometry and binary logistic regression analyses and other bioinformatics tools. Mass spectrometry (LC-HR-MS/MS) analysis identified 1,360 proteins whose expression levels were modified in CLL-derived lymphocytes. Some of these proteins were previously connected to different cancer types, including CLL, while four other highly expressed proteins were not previously reported to be associated with cancer, and here, for the first time, DDX46 and AK3 are linked to CLL. Down-regulation expression of two of these proteins resulted in cell growth inhibition. High DDX46 expression levels were associated with shorter survival of CLL patients and thus can serve as a prognosis marker. The proteins with modified expression include proteins involved in RNA splicing and translation and particularly mitochondrial proteins involved in apoptosis and metabolism. Thus, we focused on several metabolism- and apoptosis-modulating proteins, particularly on the voltage-dependent anion channel 1 (VDAC1), regulating both metabolism and apoptosis. Expression levels of Bcl-2, VDAC1, MAVS, AIF and SMAC/Diablo were markedly increased in CLL-derived lymphocytes. VDAC1 levels were highly correlated with the amount of CLL-cancerous CD19+/CD5+ cells and with the levels of all other apoptosis-modulating proteins tested. Binary logistic regression analysis demonstrated the ability to predict probability of disease with over 90% accuracy. Finally, based on the changes in the levels of several proteins in CLL patients, as revealed from LC-HR-MS/MS, we could distinguish between patients in a stable disease state and those who would be later transferred to anti-cancer treatments. The over-expressed proteins can thus serve as potential biomarkers for early diagnosis, prognosis, new targets for CLL therapy, and treatment guidance of CLL, forming the basis for personalized therapy.

The BH4 domain of anti-apoptotic Bcl-XL, but not that of the related Bcl-2, limits the voltage-dependent anion channel 1 (VDAC1)-mediated transfer of pro-apoptotic Ca2+ signals to mitochondria.

Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria.

Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein.

The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.

Misfolded mutant SOD1 directly inhibits VDAC1 conductance in a mouse model of inherited ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation-specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1(G37R). Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS.

Dominant-negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria-mediated apoptosis.

Mitochondria play a central role in the intrinsic pathway of apoptosis. Oligomerization of the mitochondrial protein VDAC1 (voltage-dependent anion channel 1) has been proposed to play a role in apoptosis in various studies. In the present study, we have generated dimeric fusion proteins consisting of tandem-linked wild-type and RuR (Ruthenium Red)-insensitive mutant VDAC1 monomers and studied the capacity of RuR to protect against apoptosis, as induced by various means. Fusion proteins composed of wild-type and/or E72Q-VDAC1 were successfully expressed in T-REx-293 cells. Bilayer-reconstituted dimeric rVDAC1 (rat VDAC1) functions as a channel-forming protein, showing typical voltage-dependence conductance, but with a unitary conductance higher than that of monomeric VDAC. As with wild-type VDAC1, overexpression of either the wild-type or mutated VDAC1 dimeric fusion protein induced apoptotic cell death. In addition, as shown previously, the anti-apoptotic effect of RuR was not observed in cells expressing E72Q-VDAC1, despite endogenous VDAC1 being present in these cells. Similar RuR insensitivity governed the VDAC1 fusion proteins comprising the E72Q mutation in either the first, second or both VDAC1 monomers of the same dimer. RuR-mediated protection against apoptosis in T-REx-293 cells, as induced by staurosporine, was observed in cells expressing VDAC1 or dimeric wild-type VDAC1. However, RuR offered no protection against staurosporine-induced apoptosis in cells expressing E72Q-VDAC1 or E72Q-containing dimeric VDAC1. These results suggest that E72Q-VDAC1 has a dominant-negative effect and implies that VDAC1 homo-oligomerization, involving intermolecular interactions, might be involved in the apoptotic process.

VDAC1 cysteine residues: topology and function in channel activity and apoptosis.

The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM (N-ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys127 and Cys232, were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)- or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC1 together with native or cysteine-less rat VDAC1, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H2O2, As2O3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.

VDAC, a multi-functional mitochondrial protein regulating cell life and death.

Research over the past decade has extended the prevailing view of the mitochondrion to include functions well beyond the generation of cellular energy. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organellar communication, aging, cell proliferation, diseases and cell death. Thus, mitochondria play a central role in the regulation of apoptosis (programmed cell death) and serve as the venue for cellular decisions leading to cell life or death. One of the mitochondrial proteins controlling cell life and death is the voltage-dependent anion channel (VDAC), also known as mitochondrial porin. VDAC, located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling cross-talk between mitochondria and the rest of the cell. VDAC is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC appears to be a convergence point for a variety of cell survival and cell death signals mediated by its association with various ligands and proteins. In this article, we review what is known about the VDAC channel in terms of its structure, relevance to ATP rationing, Ca(2+) homeostasis, protection against oxidative stress, regulation of apoptosis, involvement in several diseases and its role in the action of different drugs. In light of our recent findings and the recently solved NMR- and crystallography-based 3D structures of VDAC1, the focus of this review will be on the central role of VDAC in cell life and death, addressing VDAC function in the regulation of mitochondria-mediated apoptosis with an emphasis on structure-function relations. Understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of functions, all important for cell life and death. This review also provides insight into the potential of VDAC1 as a rational target for new therapeutics.

Voltage-dependent anion channel 1-based peptides interact with Bcl-2 to prevent antiapoptotic activity.

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.

The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins.

The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.