DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Dietary partitioning of three large carnivores in Majete Wildlife Reserve,Malawi

Between 2011 and 2012, the carnivore guild in Majete Wildlife Reserve (MWR), Malawi, was restored following the reintroduction of lion (Panthera leo) and leopard (Panthera pardus). The aim of this study was to describe and compare the diet of lion, leopard and resident spotted hyaena (Crocuta crocuta) using scat analysis. Lions and spotted hyaenas displayed the greatest dietary overlap (O_ab = 0.88) and selected mainly medium- to large-bodied prey species. Lions had a mean preferred prey weight of 120.33 ± 42.14 kg (SE), with warthog (Phacochoerus africanus) and waterbuck (Kobus ellipsiprymnus) making up 60.64% of relative biomass consumed. Spotted hyaenas had a mean preferred prey weight of 102.40 ± 41.69 kg and had a more generalised diet (B_a = 0.46) compared to lions (B_a = 0.36). In contrast, leopards occupied a dietary niche substantially lower than that of lions and spotted hyaenas, selecting relatively smaller prey with a mean preferred prey weight of 27.50 ± 6.74 kg. Our results suggest that coexistence between the resident hyaena and reintroduced lion and leopard in MWR is facilitated by dietary partitioning. We advise long-term monitoring of reintroduced carnivores in small, enclosed reserves to assess their impacts on predator and prey populations.     

Pareuchiloglanis (Teleostei: Sisoridae) from the Pearl River,China with description of three new species

We describe three new species of Pareuchiloglanis. Based on a comparison of 17 valid species of Pareuchiloglanis, the genus can be divided into two groups contingent on their gill opening size and the anus position. One group, which we call the large gill opening group, has a large gill opening extending to the base of the first pectoral-fin element; the anus is obviously closer to pelvic-fin insertion than the anal-fin origin; this group includes five species distributed in the Red and Pearl Rivers, China. The other group has a small gill opening extending only to the middle base of the pectoral-fin elements; the anus is usually located at the midpoint of the pelvic-fin insertion to the anal-fin origin or slightly behind. This group includes the other 12 species, which are distributed in the Mekong and Yangtze Rivers. The large-gill-opening group can be divided into two sub-groups based on the length of the caudal peduncle. One sub-group has a long caudal-peduncle and the distance from the anal-fin origin to caudal-fin base is greater than distance from the pelvic-fin insertion. This sub-group is only distributed in the Pearl River drainage. Another sub-group has a short caudal peduncle and the distance from the anal-fin origin to the caudal-fin base is typically smaller than the distance from the pelvic-fin insertion. This sub-group is only distributed in the Red River basin of China and Vietnam. The former will be called the large-gill-opening group with long caudal peduncle in the text and only includes one species P. longicauda. During our ongoing taxonomic work of specimens collected from Nanpan-jiang and Beipan-jiang (upper Pearl River drainage in Yunnan, China), some Pareuchiloglanis specimens that had the characters of the large-gill-opening group with long caudal peduncle represent three undescribed species.     

ARPC4 promotes bladder cancer cell invasion and is associated with lymph node metastasis

The significance of actin-related protein 2/3 complex subunit 4 (ARPC4) expression in bladder cancer, and its potential role in the invasion and migration of bladder cancer cells, has yet to be determined. This study was to identify the correlation between ARPC4 and lymph node metastasis, and to determine the role of ARPC4 in the invasive migration of T24 bladder cancer cells. One hundred and ninety-eight bladder cancer tissues and 40 normal bladder and lymph node tissues were examined. Tissue microarrays were constructed and subjected to immunohistochemical stating for ARPC4. Multiple logistic analysis was used to determine risk factors associated with bladder cancer metastasis. ARPC4 expression in T24 bladder cancer cells was suppressed using small interfering RNA and changes in protein levels were determined by Western blot analysis. The proliferation of bladder cancer cells after knocking down of ARPC4 was determined by cell counting kit-8. The effects of ARPC4 knockdown on T24 cell invasion and migration was determined using transwell and wound healing assays. Immunofluorescence analysis was performed to examine changes in pseudopodia formation and actin cytoskeleton structure. The expression of ARPC4 was elevated in bladder cancer tissues than normal tissues (84.3% vs 27.5%, P < 0.001). The multivariate logistic analysis demonstrated that the level of ARPC4, as a risk factor, was correlated with lymphatic metastasis (P < 0.05). ARPC4 knockdown attenuated proliferation, migration, invasion, and pseudopodia formation in T24 cells. ARPC4 expression, as a risk factor, is associated with lymphatic metastasis and is upregulated in bladder cancer tissues in comparison with normal tissues. Inhibition of ARPC4 expression significantly attenuates the proliferation, migration, and invasion of bladder cancer cell, possibly due to defects in pseudopodia formation.     

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T₃)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population were significantly greater in MED1^−/− cells, compared with MED1^+/+ cells. Exposure to T₃ and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.     

Cover Image,Volume 88, Issue 6

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (¹⁴²STN¹⁴⁴) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (¹⁴¹RSTN¹⁴⁴). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.