Factors that differentiate cancer risk management decisions among females with pathogenic/likely pathogenic variants in PALB2, CHEK2, and ATM.

PURPOSE: Following disclosure of pathogenic or likely pathogenic variants in hereditary cancer genes, patients face cancer risk management decisions. Through this mixed-methods study, we investigated cancer risk management decisions among females with pathogenic or likely pathogenic variants in PALB2, CHEK2, and ATM to understand why some patients follow National Comprehensive Cancer Network guidelines, whereas others do not. METHODS: Survey and interview data were cross-analyzed using a 3-stage approach. Identified factors were used to conduct coincidence analysis and differentiate between combinations of factors that result in following or not following guidelines. RESULTS: Of the 13 participants who underwent guideline inconsistent prophylactic surgery, 12 fit 1 of 3 unique patterns: (1) cancer-related anxiety in the absence of trust in care, (2) provider recommending surgery inconsistent with National Comprehensive Cancer Network guidelines, or (3) surgery occurring before genetic testing. Two unique patterns were found among 18 of 20 participants who followed guidelines: (1) anxiety along with trust in care or (2) lack of anxiety and no prophylactic surgery before testing. CONCLUSION: Health care provider recommendations and trust in care may influence whether individuals receive care that is congruent with risk levels conferred by specific genes. Interventions are needed to improve provider knowledge, patient trust in non-surgical care, and patient anxiety.

Evaluation of Genome Sequences of the Bacteriophages JeTaime and Luna22.

The mycobacteriophages JeTaime (E cluster) and Luna22 (Q cluster) were isolated from soil in Providence, Rhode Island, and Charleston, South Carolina, respectively, using a Mycobacterium smegmatis mc2 155 host. The genome of JeTaime is 75,099 bp (142 predicted genes), and that of Luna22 is 53,730 bp (87 predicted genes). Both phages exhibit Siphoviridae morphology.

Sharing genetic test results with family members of BRCA, PALB2, CHEK2, and ATM carriers.

OBJECTIVE: This study explored motivators and challenges/barriers to sharing personal genetic test results (GTR) with family members (FM). METHODS: Semi-structured, in-depth interviews were conducted with 62 women who had a pathogenic or likely pathogenic (P/LP) variant in aBRCA, PALB2, CHEK2, or ATM gene. Selective qualitative data analysis focused on eliciting motivators and challenges/barriers identified by participants when sharing their GTR with FM. RESULTS: Motivators to sharing personal GTR with FM included: health protection and prevention; moral obligation; decisional empowerment; familial ties; written resources; and contextualization for a familial cause for cancer. Challenges/barriers to family sharing included: concern for FM reactions; complexities of information; lack of closeness; perceived relevance; and emotional impact. CONCLUSIONS: All motivators and challenges/barriers were identified across BRCA and non-BRCA carriers, demonstrating commonalities in family sharing of GTR among high- to moderate-penetrance hereditary BC (breast cancer) genes. Despite challenges/barriers, participants disclosed their GTR with most close FM, yet restrictions in communication and/or strain on the timing, manner of disclosing, and strategies used varied across certain FM. PRACTICE IMPLICATIONS: These findings offer healthcare providers and researchers preliminary practical implications for broadly improving family sharing interventions across P/LP variants in BC risk genes by demonstrating important elements to include in family sharing letters.

Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into ß-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to ß-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Humanin induces conformational changes in the apoptosis regulator BAX and sequesters it into fibers, preventing mitochondrial outer-membrane permeabilization.

The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family. BCL-2-associated X apoptosis regulator (BAX) plays a pivotal role in the initiation of mitochondria-mediated apoptosis as one of the factors causing mitochondrial outer-membrane permeabilization (MOMP). Of current interest are endogenous BAX ligands that inhibit its MOMP activity. Mitochondrial-derived peptides (MDPs) are a recently identified class of mitochondrial retrograde signaling molecules and are reported to be potent apoptosis inhibitors. Among them, humanin (HN) has been shown to suppress apoptosis by inhibiting BAX translocation to the mitochondrial outer membrane, but the molecular mechanism of this interaction is unknown. Here, using recombinant protein expression, along with light-scattering, CD, and fluorescence spectroscopy, we report that HN and BAX can form fibers together in vitro Results from negative stain EM experiments suggest that BAX undergoes secondary and tertiary structural rearrangements and incorporates into the fibers, and that its membrane-associating C-terminal helix is important for the fibrillation process. Additionally, HN mutations known to alter its anti-apoptotic activity affect fiber morphology. Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis.

Promoter library designed for fine-tuned gene expression in Pichia pastoris.

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.

Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.

Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.