Elevated tumor expression of Astroprincin (FAM171A1) is an independent marker of poor prognosis in colon cancer.

BACKGROUND: Colon cancer (CC) is one of the most commonly diagnosed malignancies worldwide. Several biomarkers have been suggested for improved prognostic evaluation, but few have been implemented in clinical practice. There is a need for biomarkers that predict the tumor behavior in CC and allow stratification of patients that would benefit from adjuvant therapy. We recently identified and functionally characterized a previously unknown protein that we called ASTROPRINCIN (APCN) due to its abundance in astrocytes. APCN, also annotated as FAM171A1, is found in trophoblasts of early placenta. We demonstrated that high expression levels of APCN in cancer cells induced motility and ability of invasive growth in semisolid medium. METHODS: We screened by immunohistochemistry a tissue microarray material from the tumors of 429 CC patients with clinical follow-up in a test series and 255 CC patients in a validation series. RESULTS: We showed that low or absent APCN expression correlates with a favorable prognosis while high APCN expression was a sign of an adverse outcome. Cox uni- and multivariable analysis revealed that elevated tumor expression of APCN constitutes a robust marker of poor prognosis independent of stage, grade, patient's age, or gender. CONCLUSION: Our findings demonstrate that APCN is a novel independent prognostic marker in CC and could potentially select patients for more intense postoperative adjuvant treatment and follow-up.

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons.

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.

Astroprincin (FAM171A1, C10orf38): A Regulator of Human Cell Shape and Invasive Growth.

Our group originally found and cloned cDNA for a 98-kDa type 1 transmembrane glycoprotein of unknown function. Because of its abundant expression in astrocytes, it was called the protein astroprincin (APCN). Two thirds of the evolutionarily conserved protein is intracytoplasmic, whereas the extracellular domain carries two N-glycosidic side chains. APCN is physiologically expressed in placental trophoblasts, skeletal and hearth muscle, and kidney and pancreas. Overexpression of APCN (cDNA) in various cell lines induced sprouting of slender projections, whereas knockdown of APCN expression by siRNA caused disappearance of actin stress fibers. Immunohistochemical staining of human cancers for endogenous APCN showed elevated expression in invasive tumor cells compared with intratumoral cells. Human melanoma cells (SK-MEL-28) transfected with APCN cDNA acquired the ability of invasive growth in semisolid medium (Matrigel) not seen with control cells. A conserved carboxyterminal stretch of 21 amino acids was found to be essential for APCN to induce cell sprouting and invasive growth. Yeast two-hybrid screening revealed several interactive partners, of which ornithine decarboxylase antizyme-1, NEEP21 (NSG1), and ADAM10 were validated by coimmunoprecipitation. This is the first functional description of APCN. These data show that APCN regulates the dynamics of the actin cytoskeletal and, thereby, the cell shape and invasive growth potential of tumor cells.

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA-DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein-protein and protein-DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

Human IP3 receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons.

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.

Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment.

Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level.

Ornithine decarboxylase antizyme inhibitor 2 (AZIN2) is a signature of secretory phenotype and independent predictor of adverse prognosis in colorectal cancer.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. The two ODC antizyme inhibitors (AZIN1) and (AZIN2) are regulators of the catalytic activity of ODC. While AZIN1 is a regulator of cell proliferation, AZIN2 is involved in intracellular vesicle transport and secretion. There are no previous reports on the impact of AZIN2 expression in human cancer. We applied immunohistochemistry with antibodies to human AZIN2 on tissue micro- arrays of colorectal cancers (CRC) from 840 patients with a median follow-up of 5.1 years (range 0-25.8). The 5-year disease-specific survival rate was 58.9% (95% Cl 55.0-62.8%). High AZIN2 expression was associated with mucinous histology (p = 0.002) and location in the right hemicolon (p = 0.021). We found no association with age, gender, stage, or histological tumor grade. High tumor expression of AZIN2 predicted an unfavorable prognosis (p<0.0001, log-rank test), compared to low AZIN2 expression. Cox multivariable analysis identified AZIN2 as an independent factor of an unfavorable prognosis in CRC. The strongest AZIN2 expression was seen in invasive tumor cells having morphological features of epithelial-mesenchymal transition (EMT). Induction of EMT in HT-29 CRC cells lead to upregulated expression of endogenous AZIN2. Given that AZIN2 is a regulator of vesicle transport and secretion, we overexpressed human AZIN2 cDNA in T84 CRC cells, and found strongly enhanced accumulation of CD63-positive exosomes in the culture medium. These findings indicate that AZIN2 expression is a signature of EMT-associated secretory phenotype that is linked to an adverse prognosis in CRC.

Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues.

Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues.

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIß, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIß were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

Universal platform for quantitative analysis of DNA transposition.

BACKGROUND: Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. RESULTS: Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. CONCLUSIONS: The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.