RESUMO
When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.
Assuntos
Ribossomos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Anticorpos/metabolismo , Biocatálise , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estabilidade Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidoresRESUMO
KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for â¼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.
Assuntos
Neoplasias Colorretais/genética , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Inibidores de MTOR/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Animais , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) symptoms persist into adulthood and are associated with functional impairments. Neuroimaging studies of reward-modulated inhibitory control can identify potential objective markers of impairment and may deepen our understanding of why probands engage in costly behaviors leading to adverse outcomes. The study aimed to identify reward-modulated inhibitory control neural circuitries, their association with ADHD symptoms, and real-world implications of a decreased capacity to engage in reward-modulated inhibitory control. METHODS: A total of 106 adults (90% male) with rigorous childhood diagnoses of ADHD were scanned with functional magnetic resonance imaging during the Monetary Incentive Go/NoGo task. Adulthood symptoms of inattention and hyperactivity/impulsivity based on self- and informant report were assessed. The number of lifetime attempts taken to quit smoking were also assessed as an exemplar real-world outcome. RESULTS: Hyperactivity/impulsivity was negatively associated with activation in the pallidum and primary motor cortex when inhibiting a previously rewarded Go stimulus that yielded a small immediate reward in order to obtain a larger reward later on. Reduced recruitment of the pallidal-thalamic-motor circuit mediated the negative association between hyperactivity/impulsivity and reward-modulated inhibitory control accuracy. Reduced pallidum activation, in response to reward-modulated inhibitory control, was also associated with more attempts made to successfully quit smoking. CONCLUSIONS: Probands with persistent hyperactivity/impulsivity symptoms have alterations in brain regions that calculate the value of inhibiting an action that yields an immediate reward in order to obtain delayed larger rewards. This deficit results in poor inhibitory control on basic tasks and during real-world behaviors that rely on similar processes.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Adulto , Criança , Vias Eferentes , Feminino , Globo Pálido , Humanos , Imageamento por Ressonância Magnética , Masculino , RecompensaRESUMO
Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes.
Assuntos
Neoplasias Hepáticas/metabolismo , Animais , Proliferação de Células , Glucose/metabolismo , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutamina/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Fosfofrutoquinase-2/antagonistas & inibidores , Antineoplásicos/química , Calorimetria/métodos , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfofrutoquinase-2/química , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas , Fluxo de TrabalhoRESUMO
Translating existing and emerging knowledge of cancer biology into effective novel therapies remains a great challenge in drug discovery. A firm understanding of the target biology, confidence in the supporting preclinical research, and access to diverse chemical matter is required to lower attrition rates and prosecute targets effectively. Understanding past successes and failures will aid in refining this process to deliver further therapeutic benefit to patients. In this review, we suggest that early oncology drug discovery should focus on selection and prosecution of cancer targets with strong disease biology rather than on more chemically "druggable" targets with only modest disease-linkage. This approach offers higher potential benefit but also increases the need for innovative and alternative approaches. These include using different methods to validate novel targets and identify chemical matter, as well as raising the standards and our interpretation of the scientific literature. The combination of skills required for this emphasizes the need for broader early collaborations between academia and industry.
Assuntos
Descoberta de Drogas , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Academias e Institutos , Animais , Comportamento Cooperativo , Descoberta de Drogas/métodos , Indústria Farmacêutica , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Reprodutibilidade dos TestesRESUMO
Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT-specific multidrug sensitization is associated with enhanced autophagosome-lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2(+) breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2-dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over-expressed in HER2(+) breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy-treated patients with primary breast cancer. These data suggest that the induction of LAMP2-dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra-tumour heterogeneity.
Assuntos
Autofagia/fisiologia , Neoplasias da Mama/tratamento farmacológico , Instabilidade Cromossômica/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Ceramidas/metabolismo , Ceramidas/farmacologia , Cisplatino/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Expressão Gênica , Inativação Gênica/fisiologia , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/fisiologia , Pessoa de Meia-Idade , Moduladores de Mitose/farmacologia , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2 , Células Tumorais CultivadasRESUMO
Targeting cancer metabolism has emerged as a hot topic for drug discovery. Most cancers have a high demand for metabolic inputs (i.e. glucose/glutamine), which aid proliferation and survival. Interest in targeting cancer metabolism has been renewed in recent years with the discovery that many cancer-related (e.g. oncogenic and tumour suppressor) pathways have a profound effect on metabolism and that many tumours become dependent on specific metabolic processes. Considering the recent increase in our understanding of cancer metabolism and the increasing knowledge of the enzymes and pathways involved, the question arises: could metabolism be cancer's Achilles heel? During recent years, interest into the possible therapeutic benefit of targeting metabolic pathways in cancer has increased dramatically with academic and pharmaceutical groups actively pursuing this aspect of tumour physiology. Therefore, what has fuelled this revived interest in targeting cancer metabolism and what are the major advances and potential challenges faced in the race to develop new therapeutics in this area? This review will attempt to answer these questions by summarising recent developments in this field. We aim to illustrate why we, and others, believe that targeting metabolism in cancer presents such a promising therapeutic rationale.
Assuntos
Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , HumanosRESUMO
The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase Cgamma1 (PLCgamma1) is critical for integrin-mediated cell spreading and motility (N. Jones et al., J. Cell Sci. 118:2695-2706, 2005). In the current study we show that, on a basement membrane-type matrix, PLCgamma1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor beta-Pix; GIT1 and beta-Pix form tight complexes independently of PLCgamma1. The association of PLCgamma1 with the complex requires both GIT1 and beta-Pix and the specific array region (gammaSA) of PLCgamma1. Mutations of PLCgamma1 within the gammaSA region reveal that association with this complex is essential for the phosphorylation of PLCgamma1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either beta-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLCgamma1. Furthermore, siRNA depletion of PLCgamma1, beta-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RhoA, were able to rescue the elongation of these cells. Signaling of the PLCgamma1/GIT1/beta-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLCgamma1 with complexes containing GIT1 and beta-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLCgamma1 is also involved in the activation of Cdc42 and Rac1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfolipase C gama/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Calpaína/metabolismo , Linhagem Celular Tumoral , Forma Celular , Colágeno , Proteína Substrato Associada a Crk/metabolismo , Combinação de Medicamentos , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Laminina , Camundongos , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Proteoglicanas , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca(2+) release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely l-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was approximately 150, 60, 32, 30, and 18 min, respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca(2+) release in a human epidermoid carcinoma cell line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.
Assuntos
Artefatos , Bioensaio , Sinalização do Cálcio/efeitos dos fármacos , Meios de Cultura/química , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Linhagem Celular Tumoral , Meios de Cultura/metabolismo , Estabilidade de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Estrenos/química , Estrenos/metabolismo , Glutamina/química , Glutationa/química , Meia-Vida , Humanos , Ligação Proteica , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Soroalbumina Bovina/química , Fatores de TempoRESUMO
Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, beta, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described. These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta, or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets. Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was <6% blocked insulin-stimulated phosphorylation of endogenous GSK-3alpha and GSK-3beta isoforms by 67% and 45%, respectively, showing that GSK-3alpha and GSK-3beta are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3alpha and GSK-3beta phosphorylation, with PKBbeta having the predominant role. Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3alpha and GSK-3beta, and a pathway involving atypical PKCs, zeta/lambda, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.
Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Células Cultivadas , DNA/biossíntese , Glicogênio Sintase Quinase 3 beta , Insulina/farmacologia , Isoenzimas/metabolismo , Camundongos , Antígenos de Histocompatibilidade Menor , Sondas de Oligonucleotídeos , Fosforilação , Proteína Quinase C/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCgamma1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCgamma1 to early, integrin-regulated changes leading to cell motility. The key role of PLCgamma1 was supported by findings that specific depletion of PLCgamma1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCgamma1(-/-) cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCgamma1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCgamma1 activity, beta1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCgamma1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of beta1 integrin or Src. Our findings establish PLCgamma1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCgamma1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.
Assuntos
Movimento Celular , Integrinas/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Animais , Membrana Basal/citologia , Membrana Basal/metabolismo , Sinalização do Cálcio , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Matriz Extracelular/metabolismo , Fibroblastos , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fosfolipase C gama , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/deficiência , Fosfolipases Tipo C/genética , Quinases da Família src/metabolismoRESUMO
The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.