Structural basis for activation of Arf1 at the Golgi complex.

The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains.

Mechanisms of SSBP1 variants in mitochondrial disease: Molecular dynamics simulations reveal stable tetramers with altered DNA binding surfaces.

Several mutations in the gene for the mitochondrial single stranded DNA binding protein (SSBP1) have recently been implicated in human disease, but initial reports are insufficient to explain the molecular mechanism of disease, including the possible role of SSBP1 heterotetramers in heterozygous patients. Here we employed molecular simulations to model the dynamics of wild type and 31 variant SSBP1 tetramer systems, including 7 variant homotetramer and 24 representative heterotetramer systems. Our simulations indicate that all variants are stable and most have stronger intermonomer interactions, reduced solvent accessible surface areas, and a net loss of positive surface charge.

Consequences of compromised mitochondrial genome integrity.

Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria.

SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance.

Regulation of Arf activation occurs via distinct mechanisms at early and late Golgi compartments.

At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for trafficking from the Golgi. In , three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2.

The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase.

The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined.

In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans.

Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. Caenorhabditis elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in vivo. We report robust BER in the nuclear genome and slightly slower damage removal from the mitochondrial genome; in both cases the removal rates are comparable to those observed in mammals.

Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers.

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T.

Importance of the C2, N7, and C8 positions to the mutagenic potential of 8-Oxo-2'-deoxyguanosine with two A family polymerases.

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential.

Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek).

Purpose: Met, an oncogene product and receptor tyrosine kinase, is a keystone molecule for malignant progression in solid human tumors. We are developing Met-directed imaging and therapeutic agents, including anti-Met monoclonal antibodies (MetSeek). In this study, we compared two antibodies, Met5 and Met3, for nuclear imaging of human and canine Met-expressing tumor xenografts in nude mice.

Geldanamycins exquisitely inhibit HGF/SF-mediated tumor cell invasion.

Induction of the urokinase-type plasminogen activator (uPA) by hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in tumor cell invasion and metastasis that is mediated through the Met receptor tyrosine kinase. Geldanamycins (GA) are antitumor drugs that bind and inhibit HSP90 chaperone activity at nanomolar concentrations (nM-GAi) by preventing proper folding and functioning of certain oncoproteins. Previously, we have shown that a subset of GA derivatives exhibit exquisite potency, inhibiting HGF/SF-induced uPA-plasmin activation at femtomolar concentrations (fM-GAi) in canine MDCK cells.

Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor.

Downstream signaling that results from the interaction of hepatocyte growth factor/scatter factor (HGF/SF) with the receptor tyrosine kinase Met plays critical roles in tumor development, progression, and metastasis. This ligand-receptor pair is an attractive target for new diagnostic and therapeutic agents, preclinical development of which requires suitable animal models. The growth of heterotopic and orthotopic Met-expressing human tumor xenografts in conventional strains of immunocompromised mice inadequately replicates the paracrine stimulation by human HGF/SF (hHGF/SF) that occurs in humans with cancer.

RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival.

Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Suppression of this signaling pathway by targeting the Met protein tyrosine kinase may be an ideal strategy for suppressing malignant tumor growth. Using RNA interference technology and adenovirus vectors carrying small-interfering RNA constructs (Ad Met small-interfering RNA) directed against mouse, canine, and human Met, we can knock down c-met mRNA.

Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis.

A strict regulation of hepatocyte growth factor/scatter factor (HGF/SF)-Met signaling is essential for its appropriate function. Several negative regulators of Met signaling have been identified. Here we report that human Spry2 is induced by HGF/SF and negatively regulates HGF/SF-Met signaling.

Radioimmunoscintigraphy of human met-expressing tumor xenografts using met3, a new monoclonal antibody.

Purpose: Inappropriate expression of the receptor tyrosine kinase Met and its ligand is associated with an aggressive phenotype and poor clinical prognosis for a wide variety of solid human tumors. We are developing imaging and therapeutic agents that target this receptor-ligand complex. In this study, we evaluated the ability of radioiodinated anti-Met monoclonal antibodies from a single hybridoma clone to image human Met-expressing tumor xenografts.