Studies of the assembly pathway of human ATP synthase

The roles of the supernumerary subunits of human ATP synthase in dimerisation, oligomerisation and assembly of ATP synthase were investigated.Human mitochondrial ATP synthase is an enzyme containing 18 unlike subunits located in the inner mitochondrial membrane (IMM), where the catalytic F1 domain extends into the mitochondrial matrix and the FO domain, which contains the c8-ring rotor, the a-subunit and the supernumerary subunits, is anchored in the IMM. All the subunits, apart from the a- and A6L-subunits, are encoded in the nucleus and require transport into the mitochondria before being assembled. The a- and A6L-subunits are encoded on the mitochondrial genome. The respiratory complexes generate the proton motive force (PMF), which ATP synthase uses to generate ATP from ADP and Pi. Rotation of the α- and β-subunits with the central stalk γ-, δ- and ε-subunits is prevented by coupling the F1 domain to the FO domain via the peripheral stalk (the OSCP-, F6-, d- and b-subunits). ATP hydrolysis is prevented by the natural inhibitor of the enzyme, IF1, binding to the F1 domain. In addition to the aand, b-subunits, the FO domain contains the c8-ring and six supernumerary subunits not involved in the catalytic activity of ATP synthase. The roles of five of these subunits in the assembly of ATP synthase, the e-, f-, g-, DAPIT- and 6.8 kDa proteolipid-subunits, were investigated by suppressing or disrupting their expression individually. The e-subunit is the first of the supernumerary subunits to assemble, then the g-subunit followed by the f-, 6.8 kDa proteolipid- and DAPIT-subunits. All five supernumerary subunits investigated were required to facilitate the dimerisation and oligomerisation of ATP synthase. The e-, f- and g-subunits were found to be important for maintaining mitochondrial respiratory capacity.MR

Assembly of the membrane domain of ATP synthase in human mitochondria.

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F1-c8 complex inhibited by the ATPase inhibitor protein IF1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast Fo membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae

APOMAB®, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy

Background: Antineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMAB® representing the DAB4 clone of a La/SSB-specific murine monoclonal autoantibody is a malignant cell-death ligand, which accumulates preferentially in tumors in an antigen-specific and dose-dependent manner after DNA-damaging chemotherapy. Here, we aim to image tumor uptake of APOMAB® (DAB4) and to define its biological correlates. Methodology/Principal Findings: Brisk tumor cell apoptosis is induced in the syngeneic EL4 lymphoma model after treatment of tumor-bearing mice with DNA-damaging cyclophosphamide/etoposide chemotherapy. Tumor and normal organ accumulation of Indium 111 (111In)-labeled La-specific DAB4 mAb as whole IgG or IgG fragments was quantified by whole-body static imaging and organ assay in tumor-bearing mice. Immunohistochemical measurements of tumor caspase-3 activation and PARP-1 cleavage, which are indicators of early and late apoptosis, respectively, were correlated with tumor accumulation of DAB4. Increased tumor accumulation of DAB4 was associated directly with both the extent of chemotherapy-induced tumor cell death and DAB4 binding per dead tumor cell. Tumor DAB4 accumulation correlated with cumulative caspase-3 activation and PARP-1 cleavage as tumor biomarkers of apoptosis and was directly related to the extended median survival time of tumor-bearing mice. Conclusions/Significance: Radiolabeled La-specific monoclonal antibody, DAB4, detected dead tumor cells after chemotherapy, rather than chemosensitive normal tissues of gut and bone marrow. DAB4 identified late apoptotic tumor cells in vivo. Hence, radiolabeled DAB4 may usefully image responses to human carcinoma therapy because DAB4 would capture the protracted cell death of carcinoma. We believe that the ability of radiolabeled DAB4 to rapidly assess the apoptotic tumor response and, consequently, to potentially predict extended survival justifies its future clinical development as a radioimmunoscintigraphic agent. This article is part I of a two-part series providing proof-of-concept for the the diagnostic and therapeutic use of a La-specific monoclonal antibody, the DAB4 clone of which is represented by the registered trademark, APOMAB®.Fares Al-Ejeh, Jocelyn M. Darby, Chris Tsopelas, Douglas Smyth, Jim Manavis and Michael P. Brow

Concise review: Nanoparticles and cellular carriers-allies in cancer imaging and cellular gene therapy?

Ineffective treatment and poor patient management continue to plague the arena of clinical oncology. The crucial issues include inadequate treatment efficacy due to ineffective targeting of cancer deposits, systemic toxicities, suboptimal cancer detection and disease monitoring. This has led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable therapies. Mesenchymal stem cells (MSCs) have the intrinsic ability to “home” to growing tumors and are hypoimmunogenic. Therefore, these can be used as (a) “Trojan Horses” to deliver gene therapy directly into the tumors and (b) carriers of nanoparticles to allow cell tracking and simultaneous cancer detection. The camouflage of MSC carriers can potentially tackle the issues of safety, vector, and/or transgene immunogenicity as well as nanoparticle clearance and toxicity. The versatility of the nanotechnology platform could allow cellular tracking using single or multimodal imaging modalities. Toward that end, noninvasive magnetic resonance imaging (MRI) is fast becoming a clinical favorite, though there is scope for improvement in its accuracy and sensitivity. In that, use of superparamagnetic iron-oxide nanoparticles (SPION) as MRI contrast enhancers may be the best option for tracking therapeutic MSC. The prospects and consequences of synergistic approaches using MSC carriers, gene therapy, and SPION in developing cancer diagnostics and therapeutics are discussed. STEM CELLS 2010; 28:1686–1702

A Protocol for a Pan-Canadian Prospective Observational Study on Active Surveillance or Surgery for Very Low Risk Papillary Thyroid Cancer

BackgroundThe traditional management of papillary thyroid cancer (PTC) is thyroidectomy (total or partial removal of the thyroid). Active surveillance (AS) may be considered as an alternative option for small, low risk PTC. AS involves close follow-up (including regularly scheduled clinical and radiological assessments), with the intention of intervening with surgery for disease progression or patient preference.MethodsThis is a protocol for a prospective, observational, long-term follow-up multi-centre Canadian cohort study. Consenting eligible adults with small, low risk PTC (< 2cm in maximal diameter, confined to the thyroid, and not immediately adjacent to critical structures in the neck) are offered the choice of AS or surgery for management of PTC. Patient participants are free to choose either option (AS or surgery) and the disease management course is thus not assigned by the investigators. Surgery is provided as usual care by a surgeon in an institution of the patient’s choice. Our primary objective is to determine the rate of ‘failure’ of disease management in respective AS and surgical arms as defined by: i) AS arm – surgery for progression of PTC, and ii) surgical arm - surgery or other treatment for disease persistence or progression after completing initial treatment. Secondary outcomes include long-term thyroid oncologic and treatment outcomes, as well as patient-reported outcomes.DiscussionThe results from this study will provide long-term clinical and patient reported outcome evidence regarding active surveillance or immediate surgery for management of small, low risk PTC. This will inform future clinical trials in disease management of small, low risk papillary thyroid cancer.Registration detailsThis prospective observational cohort study is registered on clinicaltrials.gov (NCT04624477), but it should not be considered a clinical trial as there is no assigned intervention and patients are free to choose either AS or surgery

Variable cardiovascular phenotypes associated with SMAD2 pathogenic variants

SMAD2 is a downstream effector in the TGF-beta signaling pathway, which is important for pattern formation and tissue differentiation. Pathogenic variants in SMAD2 have been reported in association with arterial aneurysms and dissections and in large cohorts of subjects with complex congenital heart disease (CHD). We used whole exome sequencing (WES) to investigate the molecular cause of CHD and other congenital anomalies in three probands and of an arterial aneurysm in an additional patient. Patients 1 and 2 presented with complex CHD, developmental delay, seizures, dysmorphic features, short stature, and poor weight gain. Patient 3 was a fetus with complex CHD and heterotaxy. The fourth patient is an adult female with aortic root aneurysm and physical features suggestive of a connective tissue disorder. WES identified pathogenic truncating variants, a splice variant, and a predicted deleterious missense variant in SMAD2. We compare the phenotypes and genotypes in our patients with previously reported cases. Our data suggest two distinct phenotypes associated with pathogenic variants in SMAD2: complex CHD with or without laterality defects and other congenital anomalies, and a late-onset vascular phenotype characterized by arterial aneurysms with connective tissue abnormalities

Needs assessment for a decision support tool in oral cancer requiring major resection and reconstruction: a mixed-methods study protocol

Introduction Advanced oral cancer and its ensuing treatment engenders significant morbidity and mortality. Patients are often elderly with significant comorbidities. Toxicities associated with surgical resection can be devastating and they are often highlighted by patients as impactful. Given the potential for suboptimal oncological and functional outcomes in this vulnerable patient population, promotion and performance of shared decision making (SDM) is crucial.Decision aids (DAs) are useful instruments for facilitating the SDM process by presenting patients with up-to-date evidence regarding risks, benefits and the possible postoperative course. Importantly, DAs also help elicit and clarify patient values and preferences. The use of DAs in cancer treatment has been shown to reduce decisional conflict and increase SDM. No DAs for oral cavity cancer have yet been developed.This study endeavours to answer the question: Is there a patient or surgeon driven need for development and implementation of a DA for adult patients considering major surgery for oral cancer?Methods and analysis This study is the first step in a multiphase investigation of SDM during major head and neck surgery. It is a multi-institutional convergent parallel mixed-methods needs assessment study. Patients and surgeon dyads will be recruited to complete questionnaires related to their perception of the SDM process (nine-item Shared Decision-Making Questionnaire, SDM-Q-9 and SDM-Q-Doc) and to take part in semistructured interviews. Patients will also complete questionnaires examining decisional self-efficacy (Ottawa Decision Self-Efficacy Scale) and decisional conflict (Decisional Conflict Scale). Questionnaires will be completed at time of recruitment and will be used to assess the current level of SDM, self-efficacy and conflict in this setting. Thematic analysis will be used to analyse transcripts of interviews. Quantitative and qualitative components of the study will be integrated through triangulation, with matrix developed to promote visualisation of the data.Ethics and Dissemination This study has been approved by the research ethics boards of the Nova Scotia Health Authority (Halifax, Nova Scotia) and the University Health Network (Toronto, Ontario). Dissemination to clinicians will be through traditional approaches and creation of a head and neck cancer SDM website. Dissemination to patients will include a section within the website, patient advocacy groups and postings within clinical environments