N-terminal Mutant Research Articles

Two rare variants of the ANXA11 gene identified in Chinese patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. A recent study has identified mutations in the ANXA11 gene (encoding the calcium-binding protein annexin A11) associated with ALS. Mutation screening of ANXA11 protein-coding exons was performed in a Chinese cohort of 434 patients with sporadic ALS and 50 index patients with familial ALS. Polymerase chain reaction and Sanger sequencing were used for mutation detection. We failed to discover an N-terminal mutation, which was common in the Caucasian cohort. We revealed two rare heterozygous missense variants, c.878C>T (p.A293V) and c.921C>G (p.I307M), which are absent from the population databases and non-neurological controls. They are both located in the conserved annexin domain. The carriers of the mutation exhibited the classical ALS phenotype without cognitive impairment. Our results suggested that further functional studies for these variants are required to support the pathogenicity.

3026 - Bi-Allelic CEBPA Mutation Combined with a Novel GATA2 Mutation Develops an Acute Erythroid Leukemia Through a Bi-Potent Leukemic Initiating Population

Acute erythroid leukemia (AEL) is a subtype of acute myeloid leukemia (AML) representing <5% of AML cases. There are two subtypes of AEL: pure erythroid leukemia and erythroleukemia (erythroid/myeloid leukemia). Sequencing studies has comprehensively characterized the mutational landscape of AML identifying mutations that are significantly associated such as the bi-allelic CEBPA and GATA2 mutations (Papaemmanuil et al.; 2016). Recently, sequencing AEL patients also found a high frequency of bi-allelic CEBPA and GATA2 co-mutated (Ping et al.; 2016). As there are currently no models of AEL we combined three knock-in mouse strains, a novel Gata2 point mutation in the N-terminal zinc finger crossed with a knock-in of the N-terminal Cebpa mutation, and a C-terminal Cebpa mutation to generate CebpaN/C;Gata2+/+ (NC genotype) and CebpaN/C;Gata2G320D/ + (NCG genotype). To bypass Cebpa mutation perinatal lethality we performed competitive transplantations using mutant fetal liver cells and wild type bone marrow (BM) competitor cells. By analyzing myeloid reconstitution over time in the peripheral blood we found the myeloid differentiation block caused by the N-terminal mutation in NC mice was prevented when co-expressed with Gata2G320D. This correlated with a more aggressive disease in NCG compared to NC transplanted mice. 100% of NC mice developed an AML while 50% of NCG mice developed an AML, and the remaining 50% developed an AEL of the erythroleukemia subtype, identified by the presence of both myeloblasts and erythroblasts in the BM, spleen and peripheral blood. NCG AEL mice had a significantly shorter overall survival than NCG AML mice. Secondary transplant of different sorted BM populations from NCG AML or NCG AEL mice showed that Lin-cKit+FcR+ GMP-like cells could propagate and fully replicate the two diseases, identifying the putative leukemic initiating cell (LIC) population. Together our study provides a novel model of the erythroleukemia subtype of AEL where both the myeloblasts and erythroblasts develop through a bi-potent LIC population.

Natural and targeted isovariants of the rice actin depolymerizing factor 2 can alter its functional and regulatory binding properties

Actin depolymerizing factors (ADFs) are ubiquitous actin-binding proteins that play essential roles in maintaining cellular actin dynamics by depolymerizing/severing F-actin. Plant ADF isoforms show functional divergence via differential biochemical and cellular properties. We have shown previously that ADF2 of rice (OsADF2) and smooth cordgrass (SaADF2) displayed contrasting biochemical properties and stress response in planta. As a proof-of-concept that amino acid variances contribute to such functional difference, single amino acid mutants of OsADF2 were generated based on its sequence differences with SaADF2. Biochemical studies showed that the single-site amino acid mutations altered actin binding, depolymerizing, and severing properties of OsADF2. Phosphosensitive mutations, such as serine-6>threonine, changed the regulatory phosphorylation efficiency of ADF2 variants. The N-terminal mutations had greater effect on the phosphorylation pattern of OsADF2, whereas C-terminal mutations affected actin binding and severing. The presence of introduced mutations in isovariants of monocot ADF suggests that these residues are significant control points regulating their functional divergence, including abiotic stress response.

Two Strategies of Pseudomonas syringae to Avoid Recognition of the HopQ1 Effector in Nicotiana Species.

Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant – microbe interactions.

Abstract 2307: Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer

Abstract Introduction Approximately 15% of muscle invasive bladder cancers harbor mutations in the CDKN1A gene, which encodes the p53 target gene p21, with the majority of alterations truncating the peptide. We previously found that proximal truncation of p21 sensitized cells to cisplatin, while distal truncations made them more resistant. We hypothesized that truncations preserving the CDKi domain but ablating the PCNA binding domain would abrogate p21's ability to sequester PCNA but maintain cell cycle arrest capacities, enhancing the interaction between mono-ubiquitiniated-PCNA (ub-PCNA) and DNA polymerase eta (Pol η), resulting in increased TLS and DNA damage tolerance. Methods We used SW780 cell line for this study based on its resistance to cisplatin and p53 and p21 WT status. CRISPR sgRNAs designed to target amino acids 12 and 109 (SW780-sg12 and SW780-sg109) were used to introduce frameshifts and disrupt the majority of the peptide or only the PCNA the binding domain, respectively. sgRNA designed to target GFP was used as a control (sgGFP). We treated the cells with GI50 dose of cisplatin and analyzed post-cisplatin cell-cycle profile over 96 hours using PI-FACS and immunoblot. We treated all three cell types with increasing doses (0-40 μM) of cisplatin for 24 hours and measured the levels of ub-PCNA (K164) and Pol η using immunoblot. Results SW780-sgGFP arrested in S-phase at 48 and 72 hours but overcame this arrest at 96 hours and began to cycle again. SW780-sg12 were unable to arrest in S-phase, whereas SW780-sg109 were able to arrest in S-phase at 48 to 96 hours. This pattern of S-phase arrest was further confirmed by prolonged upregulation of cyclin D1 in SW780-sg109 and brief upregulation in SW780-sgGFP. ub-PCNA and Pol η accumulated in a dose responsive manner in all three cell types at 24 hours after cisplatin treatment. ub-PCNA increased abruptly at higher cisplatin doses in SW780-sgGFP, whereas the increase was gradual in CDKN1A-edited cells. Interestingly, in SW780-sg109, Pol η induction was strongest at all doses. Conclusion Cisplatin causes S-phase arrest in SW780-sgGFP cells, possibly by inducing replicative stress. However, this arrest is eventually bypassed and the cells progress through the cell cycle. S-phase arrest is not possible in the absence of a functioning p21, indicating that p21 plays a vital role in bringing about post-cisplatin cell cycle arrest. Interestingly, truncated p21 with intact CDKi domain is capable of causing stronger and more prolonged S-phase arrest compared to the full-length peptide. TLS may play a role in DNA damage tolerance in SW780 cells as indicated by elevated ub-PCNA and pol η. Stronger accumulation of Pol η in SW780-sg109, possibly enhances TLS and thus cisplatin resistance. Moving forward, we would like to confirm the anticipated competition between p21 and Pol η for PCNA and further verify this phenomenon in other bladder cancer cell lines. Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2307.

Characterization of an N-terminal mutant of αA-crystallin αA–R21Q associated with congenital cataract

Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a “mutational hotspot,” with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin — αA–R21L and αA–R21W have been associated with congenital cataract. A third mutant of R21, αA–R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA–R21Q mutation on αA-crystallin function, recombinant αA–R21Q was expressed, purified and characterized in vitro. Compared to wild-type αA-crystallin, the recombinant αA–R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA–R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA–R21Q homo-oligomers and hetero-oligomers also prevented H2O2–induced apoptosis in ARPE–19 cells. Taken together, αA–R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.

De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder

Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non-truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiquitination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.

Deciphering the Role of SLX4IP in Telomere Maintenance Mechanisms

Telomeres are protective caps found at chromosomal ends that act to prevent attrition of the genome and aberrant activation of DNA repair mechanisms. In somatic cells, telomeric DNA progressively shortens with rounds of replication until a critically short length is reached triggering the cell to undergo senescence to prevent loss of genomic information. However, some cells express telomerase to elongate telomeres and avoid reaching this critical length, thus instilling additional replicative capacity. Notably, 85% of all cancer cells upregulate telomerase and the remaining 15% primarily utilize the alternative lengthening of telomeres (ALT) pathway which relies upon homologous recombination. There is evidence to suggest that cancer cells have the ability to switch to the ALT pathway, should telomerase activity be compromised, but the molecular switch regulating this process is unknown. A recently identified protein, SLX4IP, appears to play a critical role in telomere homeostasis and the telomerase‐to‐‐ALT conversion. In order to identify the basic function of SLX4IP in telomere homeostasis, the interactome of SLX4IP must be interrogated to identify additional proteins involved under specific conditions. For example, identifying differences between the interactomes in telomerase‐positive and ALT‐positive cells will provide insight regarding SLX4IP in telomere maintenance mechanisms. Initially, immunoprecipitation experiments were carried out to identify discrepancies in the proportion of known SLX4IP binding partners among telomerase‐positive and ALT‐positive cell lines. As expected, SLX4IP interacts with TRF2, a telomeric binding protein, and SLX4, an ALT‐associated nuclease scaffold protein, but with varying degrees among the cell types tested. To further dissect the interactome of SLX4IP, wild type SLX4IP, an N‐terminal mutant being interrogated for structural studies, and a C‐terminal mutant which represents a deletion mutant identified in acute lymphoblastic leukemia patients exhibiting poor prognosis were stably overexpressed in telomerase‐positive and ALT‐positive cell lines followed by subsequent immunoprecipitation and proteomic evaluation. This evaluation will provide valuable information as to which regions of SLX4IP are responsible for orchestrating particular interactions in each cell type. With this knowledge, a greater understanding of SLX4IP's role in telomere homeostasis and the telomerase‐to‐ALT conversion can be elucidated.Support or Funding InformationThis work was supported in part by an NIH T32 (GM008803) grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NAD+ Bound N‐terminal Domain of CARDS Toxin Increases IL‐1β Secretion

Mycoplasma pneumoniae (Mp) is an atypical bacterium that is linked to various respiratory diseases such as walking pneumonia and asthma. Upon infection, Mp produces a 591‐aa virulence factor known as Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX). The crystal structure of this 591‐aa cytotoxin reveals a triangular molecule comprised of an N‐terminal ADP‐ribosylating domain and a C‐terminal tandem β‐trefoil domain that is responsible for vacuolation of the host's cells. Based on structural and sequence homology to other ADP ribosylating toxins, the NAD+ binding site has been predicted, yet none of the published structures of the CARDS toxin contain NAD+. The loop connecting the N− and the C‐termini is anchored using a disulfide bond that surrounds the NAD+ binding site which might sterically hinder NAD+ binding. An N‐terminal truncation mutant (NTTM) lacking the disulfide bond has been cloned and the protein was expressed, purified and crystallization trials are underway. The NTTM was also used to treat U937 cells in presence and absence of NAD+ to test the effect of the NTTM on cytokine secretion. The data show that NTTM causes an increase in the secretion of IL‐1β and TNF‐α in presence of NAD+ suggesting an increase in the inflammatory response to the NTTM in presence of NAD+.Support or Funding InformationSt. Mary's University Biaggini Funds and The San Antonio Livestock Expedition FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutant huntingtin affects p53 function in a context‐dependent manner in a cell model of Huntington's disease

Huntington's disease (HD) is an adult‐onset neurodegenerative disease caused by expansion of a polyglutamine (polyQ) repeat in the N‐terminal region of the huntingtin (htt) protein. In HD, multiple N‐terminal mutant htt fragments, smaller than the first 500 amino acids, have been found to accumulate in disease tissue. These N‐terminal htt fragments associate abnormally with many cellular proteins, disturbing their function. The p53 tumor suppressor protein has been shown to abnormally interact with mutant htt and is associated with cellular dysfunction in HD. However, it is unknown whether different htt fragments affect p53 function differentially. Here, we investigated the affect of different length N‐terminal mutant htt fragments on p53 localization and function in a human cell model of HD. We found that shorter htt fragments cause more accumulation of p53 protein in the nucleus which is associated with decreased cell viability. Our results demonstrate the importance of mutant htt protein context in HD. While the p53 pathway is implicated as a potential target to ameliorate Huntington disease progression, the therapeutic benefit may be limited by the relative abundance of shorter N‐terminal htt fragments in target cells.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. 1458212. JC was supported in part by a faculty fellowship from the Appalachian College Association.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of N-terminal Mutations on β-amyloid Peptide Aggregation and Toxicity

Human amyloid β1–42 (hAβ1–42) peptides are known to self-aggregate into oligomers that contribute to the degeneration of neurons and development of Alzheimer's disease (AD) pathology. Unlike humans, rodents do not develop AD, possibly due to differences in three amino acids (R5G, Y10F and H13R) within the hydrophilic N-terminal domain of Aβ1–42. This is partly supported by evidence that hAβ1–42 is more prone to fibrillization and has a higher cellular toxicity than rodent Aβ1–42 (rAβ1–42). Mutagenesis studies, however, have shown that correlation between fibrillization potential and toxicity is not always direct. Thus, to understand better how N-terminal mutations can affect hAβ1–42 toxicity through oligomerization, we evaluated fibrillization kinetics, oligomer sizes and toxicity profiles of double mutant (human toward rodent) Aβ1–42. Additionally, we tested the mutant peptides in combination with hAβ1–42, to assess effects on hAβ1–42 aggregation/toxicity. Our results clearly show that double mutations to humanize rAβ1–42 result in a significantly reduced efficiency of fibril formation, as determined by Thioflavin-T aggregation assays and confirmed with electron micrographic studies. Interestingly, the mutants are still able to aggregate into oligomers, which are predominantly larger than those comprised of hAβ1–42. Our cell viability experiments further showed a rank order of oligomer toxicity of hAβ1–42 > rAβ1–42 ≫ mutant Aβ1–42, suggesting that toxicity can be influenced by N-terminal Aβ1–42 mutations via reduction of fibril formation and/or alteration of oligomer size. These results, taken together, confirm that N-terminal mutations can affect Aβ fibril and oligomer formation with reduced toxicity despite lying outside the core amyloid region of Aβ peptide.

A Filter Retardation Assay Facilitates the Detection and Quantification of Heat-Stable, Amyloidogenic Mutant Huntingtin Aggregates in Complex Biosamples.

N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.

Mutations outside the N-terminal part of RBCK1 may cause polyglucosan body myopathy with immunological dysfunction: expanding the genotype\u2013phenotype spectrum

A subset of patients with polyglucosan body myopathy was found to have underlying mutations in the RBCK1 gene. Affected patients may display diverse symptoms ranging from skeletal muscular weakness, cardiomyopathy to chronic autoinflammation and immunodeficiency. It was suggested that the exact localization of the mutation within the gene might be responsible for the specific phenotype, with N-terminal mutations causing severe immunological dysfunction and mutations in the middle or C-terminal part leading to a myopathy phenotype. We report the clinical, immunological and genetic findings of two unrelated individuals suffering from a childhood-onset RBCK1-asscociated disease caused by the same homozygous truncating mutation (NM_031229.2:c.896_899del, p.Glu299Valfs*46) in the middle part of the RBCK1 gene. Our patients suffered from a myopathy with cardiac involvement, but in contrast to previous reports on mutations in this part of the gene, also displayed signs of autoinflammation and immunodeficiency. Our report suggests that RBCK1 mutations at locations that were previously thought to lack immunological features may also present with immunological dysfunction later in the disease course. This notably broadens the genotype–phenotype correlation of RBCK1-related polyglucosan body myopathy.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Variable outcome and methylation status according to CEBPA mutant type in double-mutated acute myeloid leukemia patients and the possible implications for treatment.

Although CEBPA double-mutated (CEBPADM) acute myeloid leukemia is considered to be a favorable-risk disease, relapse remains a major cause of treatment failure. Most CEBPADM patients have a classic biallelic mutant combination with an N-terminal mutation leading to production of p30 protein plus a C-terminal loss-of-function in-frame indel mutation (CEBPAClassic-DM), but approximately one-third of cases have one or more non-classic mutations, with diverse combinations reported, and there is little information on the consequences of such mutants. We evaluated outcome in a cohort of 104 CEBPADM patients, 79 CEBPAClassic-DM and 25 with non-classic mutants, and found that the latter may have poorer survival (5-year overall survival 64% vs. 46%; P=0.05), particularly post relapse (41% vs. 0%; P=0.02). However, for this analysis, all non-classic cases were grouped together, irrespective of mutant combination. As CEBPADM cases have been reported to be hypermethylated, we used methylation profiling to assess whether this could segregate the different mutants. We developed a CEBPAClassic-DM methylation signature from a preliminary cohort of 10 CEBPADM (including 8 CEBPAClassic-DM) and 30 CEBPA wild-type (CEBPAWT) samples, and independently validated the signature in 17 CEBPAClassic-DM cases. Assessment of the signature in 16 CEBPADM cases with different non-classic mutant combinations showed that only 31% had a methylation profile equivalent to CEBPAClassic-DM whereas for 69% the profile was either intermediate between CEBPAClassic-DM and CEBPAWT or equivalent to CEBPAWT. These results suggest that CEBPADM cases with non-classic mutants may be functionally different from those with CEBPAClassic-DM mutants, and should not automatically be included in the same prognostic group. (AML12 is registered under ISRCTN17833622 and AML15 under ISRCTN17161961).

The N-terminal pY33XL motif of CaPsy2 is critical for the function of protein phosphatase 4 in CaRad53 deactivation, DNA damage-induced filamentation and virulence in Candida albicans

Protein phosphatase PP4 is composed of one catalytic subunit and one or two regulatory subunits and conserved in eukaryotic cells. The catalytic subunit CaPph3 forms a complex with the regulatory subunit CaPsy2, which dephosphorylates activated CaRad53 during adaptation to and recovery from MMS-mediated DNA damage. We show here that the N-terminal Y33A mutation of CaPsy2 blocks the interaction between CaPph3 and CaRad53, the deactivation of CaRad53 and the morphologic switch in recovery from genotoxic stress. In Saccharomyces cerevisiae, the ScPph3-ScPsy2-ScPsy4 complex functions to dephosphorylate γH2A. In this study, we show that CaPsy4 is a functional homolog of ScPsy4 and not involved in the deactivation of CaRad53 or CaHta, the ortholog of H2A. However, deletion of CaPSY4 causes C. albicans cells a sensitivity to genotoxic reagents and a defect in DNA damage-induced filamentation. CaPsy4 interacts with both CaPph3 and CaPsy2, but the function of CaPsy4 is independent of CaPph3 and CaPsy2 in response to genotoxic stress. C. albicans cells lacking CaPPH3, CaPSY2 or CaPSY4, and C. albicans cells carrying the Y33A mutation of CaPSY2, show increased virulence to mice. Therefore, PP4 plays a negative role in regulating the DNA damage-induced filamentation and the virulence in C. albicans.

Processing of OPA1 with a novel N-terminal mutation in patients with autosomal dominant optic atrophy: Escape from nonsense-mediated decay.

Autosomal Dominant Optic Atrophy (ADOA) is the most common dominantly inherited optic neuropathy. In the majority of patients it is caused by OPA1 mutations and those predicted to introduce a premature termination codon (PTC) are frequently detected. Transcripts containing PTC may be degraded by nonsense-mediated mRNA decay (NMD), however very little is known about an effect of OPA1 mutations on NMD activation. Here, using a combination of linkage analysis and DNA sequencing, we have identified a novel c.91C>T OPA1 mutation with a putative premature stop codon (Q31*), which segregated with ADOA in two Polish families. At the mRNA level we found no changes in the amount of OPA1 transcript among mutation carriers vs. non-carriers. Specific allele quantification revealed a considerable level of the OPA1 mutant transcript. Our study identifies a novel pathogenic OPA1 mutation and shows that it is located in the transcript region not prone for NMD activation. The data emphasizes the importance of analyzing how mutated genes are being processed in the cell. This gives an insight into the molecular mechanism of a genetic disease and promotes development of innovative therapeutic approaches.

HSP70-Hrd1 axis precludes the oncorepressor potential of N-terminal misfolded Blimp-1s in lymphoma cells

B lymphocyte-induced maturation protein-1 (Blimp-1) ensures B-cell differentiation into the plasma cell stage, and its instability constitutes a crucial oncogenic element in certain aggressive cases of activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). However, the underlying degradation mechanisms and their possible therapeutic relevance remain unexplored. Here, we show that N-terminal misfolding mutations in ABC-DLBCL render Blimp-1 protein susceptible to proteasome-mediated degradation but spare its transcription-regulating activity. Mechanistically, whereas wild-type Blimp-1 metabolism is triggered in the nucleus through PML-mediated sumoylation, the degradation of lymphoma-associated mutants is accelerated by subversion of this pathway to Hrd1-mediated cytoplasmic sequestration and ubiquitination. Screening experiments identifies the heat shock protein 70 (HSP70) that selects Blimp-1 mutants for Hrd1 association, and HSP70 inhibition restores their nuclear accumulation and oncorepressor activities without disrupting normal B-cell maturation. Therefore, HSP70-Hrd1 axis represents a potential therapeutic target for restoring the oncorepressor activity of unstable lymphoma-associated Blimp-1 mutants.

The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the “turn” (Ser644) and “hydrophobic” (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2-dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1.