Differential expression of ankyrin repeat domain 29 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding ankyrin repeat domain 29, ANKRD29, when comparing primary tumors of the breast to the tissue of origin, the normal breast. ANKRD29 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of ANKRD29 in primary tumors of the breast was correlated with overall survival in patients with luminal A and luminal B subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by molecular subtype. ANKRD29 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13–24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

ANKRD11 is differentially expressed in brain metastatic human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with brain metastasis in humans with metastatic breast cancer. We found that ankyrin repeat domain 11, encoded by ANKRD11, was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. ANKRD11 mRNA was present at decreased quantities in brain metastatic tissues as compared to primary tumors of the breast. Importantly, expression of ANKRD11 in primary tumors was significantly correlated with patient overall survival. Modulation of ANKRD11 expression may be relevant to the biology by which tumor cells metastasize from the breast to the brain in humans with metastatic breast cancer.

Determination of Ankyrin Repeats Domain (ANK) a of RNASE L Gene in Hepatitis C Patients and its effects on Viral Load

Background: Hepatitis C virus (HCV) is one of the major global causes of death. Different types of gene are involved as Ankyrin repeat domains of RNASE L gene. It performs a significant role in antiviral response, regulated by interferon, and involved in cleavage of RNA. Therefore, aim of this study was to identify Ankyrin repeat domain expression in Hepatitis C positive patients and correlate it with viral load of Hepatitis C. Methods: In this study, a total of 80 HCV positive patient’s whole blood samples were investigated. RNA was extracted from plasma followed by Real Time PCR for quantization of HCV viral load and genotypic analyses. DNA was also extracted from these samples followed by PCR amplification of Ankyrin repeat domain of RNASE L gene. Data was analyzed using SPSS Results: All of the patients (n=80) included in study had HCV infection. Mean age of patients was 50.86 ±14.84 years. Among them, 48(63.8%) were males and 32 (36.1%) were females. Majority of patients were males and belonged to age group 58-73 years age. All HCV infected individuals 36 (45%) had HCV genotype 3 and had viral loads mean range 837404.21 ±1302. Therefore, Ankyrin repeats domain of RNASE L gene expression was high in HCV patients sample with viral load of 17.00±15.1. Conclusion: Ankyrin repeat domain expression was observed in Hepatitis C patients and its significant correlation with viral load of Hepatitis C. Ankyrin repeat domain of RNASE L gene in conjunction with therapeutic intervention are required for establishing better strategies for controlling HCV infection.

Skd3 (human ClpB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations

Cells have evolved specialized protein disaggregases to reverse toxic protein aggregation and restore protein functionality. In nonmetazoan eukaryotes, the AAA+ disaggregase Hsp78 resolubilizes and reactivates proteins in mitochondria. Curiously, metazoa lack Hsp78. Hence, whether metazoan mitochondria reactivate aggregated proteins is unknown. Here, we establish that a mitochondrial AAA+ protein, Skd3 (human ClpB), couples ATP hydrolysis to protein disaggregation and reactivation. The Skd3 ankyrin-repeat domain combines with conserved AAA+ elements to enable stand-alone disaggregase activity. A mitochondrial inner-membrane protease, PARL, removes an autoinhibitory peptide from Skd3 to greatly enhance disaggregase activity. Indeed, PARL-activated Skd3 solubilizes α-synuclein fibrils connected to Parkinson’s disease. Human cells lacking Skd3 exhibit reduced solubility of various mitochondrial proteins, including anti-apoptotic Hax1. Importantly, Skd3 variants linked to 3-methylglutaconic aciduria, a severe mitochondrial disorder, display diminished disaggregase activity (but not always reduced ATPase activity), which predicts disease severity. Thus, Skd3 is a potent protein disaggregase critical for human health.