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Background Several hundred million of the 3 billion formally employed people worldwide are at risk from high levels of solar ultraviolet radiation (UVR). Chronic light damage to the skin can lead to nonmelanoma skin cancer (NMSC), especially when irradiation is too high and is acquired for decades. However, data with uniform metrics, high resolution over time and in-depth occupational profiles are not available. Objectives To build a worldwide usable matrix of UVR exposure in occupations and to show use cases for the data. Methods One thousand test persons were recruited to wear electronic data logger dosimeters during their working time for 7 months each. The measurements yielded 3.7 billion data points for around 48 000 days with high-quality data capture covering more than 250 occupations and 650 activities. Scientific evaluation of the data included daily and half-hourly means, geographical transfer calculations to the world, threshold exceedance quotas, transcriptome effects, and occupational disease estimates. Results A compendium for global use is presented. In-depth analyses and the resulting implications for research have been elaborated to directly link exposure data to effects in the human body. Interestingly, the annual irradiances of the different occupations span a wide range of values: from about 650 to 50 standard erythemal doses, with different distributions over the months. Detailed exposure data per occupation were derived, and the risk on an occupational or activity basis assuming different exposure level quotas was quantified. This showed that, for example, in temperate latitudes, all activities with a duration of > 2 h outdoors are associated with increased NMSC risk. Conclusions We offer our work to enable sound studies on the nature of ultraviolet-induced skin cancer, dose–response relationships, intermittency of skin exposure, and derivation of limit values. Sociological studies on prevention are now possible. Practitioners may use the findings for their daily work with employees.

Objectives. Non-melanoma skin cancer (NMSC) is the most common human malignancy and is strongly related to solar ultraviolet (UV) radiation exposure. Occupational UV exposure is associated with a two-fold increased risk of developing NMSC, but still only few European countries recognize NMSC as an occupational disease. The study aims to analyze the clinical and histopathological features of NMSC with regard to personal cumulative occupational UV exposure doses. Material and methods. A retrospective, analytical, comparative study was conducted during 2017-2019 with focus on outdoor workers presenting with NMSC. Occupational solar UV exposure dose was calculated based on the Wittlich algorithm and cumulative lifetime exposure of the general population of the same age was estimated according to German Standards of 130 SED/year. Subjects were divided in two groups (OW1 and OW2) according to differences between occupational exposure and estimated cumulative lifetime exposure of the general population. High-risk clinical and histopathological characteristics were compared between groups. Outcomes. The study included 25 consecutive outdoor workers diagnosed with BCC (N=21) and SCC (N=4). Occupational solar UV exposure doses were calculated for Romanian workers and identified mean values of 17695 SED two times higher than estimative leisure cumulative lifetime solar UV exposure for a person of the same age from the general population according to German standards of 130 SED/year. Subjects were classified in two groups: group 1 (OW1) included subjects who were exposed to an additional occupational UV dose less than 50% of the cumulative lifetime exposure dose of a person of the same age from the general population according to German standards and group 2 (OW2) included subjects exposed to additional professional UV doses higher than 50%. 12 subjects were included in OW 1 and 13 subjects in OW2. Data analysis between groups identified significant statistical differences with regard to high-risk body locations and tumour size, present in 100% of OW2 and 58.3% of OW1 (χ2 (1, N=51) = 6.771, p=0.015) and to high-risk histopathological subtypes, present in 56% OW2 and 33.3% OW1 (χ2 (1, N=51) = 4.812, p=0.036). Ulceration was present on the histopathological report of all subjects in OW2. OW2 was associated with higher percentages of moderate to severe grades of actinic elastosis and of desmoplastic reaction and a more rapid evolution of lesions. Conclusions: Outdoor workers in Romania are exposed to an occupational solar UV dose two times higher than the cumulative lifetime exposure dose of the general population of the same age which may receive up to 130 SED/year according to German standards and they bear an increased risk of NMSC. Occupational NMSC induced by UV exposure should be recognized as an occupational disease if the subject received more than 50% additional UV exposure at the workplace.

Non-melanoma skin cancer (NMSC) is the most common cancer in western countries. Legislative bodies and stakeholders like WHO and EU strongly promote protection against solar UVR, especially in workers. Occupational health prevention must be introduced as a strong instrument in workers protection also with regard to occupational disease issues. To date, criteria for both occupational health prevention and occupational disease are missing and the identification of risk groups has no metric basis. Here I report a criteria analysis based on the largest comprehensive data set of occupational ultraviolet radiation exposure of outdoor workers. With detailed research on occupation-specific dosimetric measurements of 45.000 measurement days in 176 occupations and sub-occupations, it is possible to map criteria for occupational health prevention specifically and to identify affected occupations. The number of employees affected can be elucidated worldwide. For the first time, a direct link to retrospective occupational disease criteria could be established. Of the 176 occupations and sub-occupations selected for this work, 153 (=87%) exceed the criterion for occupational health prevention and thus need special attention. This includes all occupations with annual exposures of more than 150 SED. Employment figures for the EU and the world yield the total number of affected workers to be 36.1 million and more than 500 million, respectively. These new criteria for occupational health prevention are valid and in good agreement with international research on limit values by WHO and ICNIRP. If applied correctly and consistently, these criteria can prevent occupational disease. It will be possible to identify occupations and sub-occupations that have an urgent need for prevention to avoid chronic skin damage leading to cancer. This research serves as a basis for policy making and clinical risk identification, as well as for daily practice of occupational physicians and employers responsible for risk assesment.

(1) Measuring personal exposure to solar ultraviolet radiation (UVR) poses a major challenges for researchers. Often, the study design determines the measuring devices that can be used, be it the duration of measurements or size restrictions on different body parts. It is therefore of great importance that measuring devices produce comparable results despite technical differences and modes of operation. Particularly when measurement results from different studies dealing with personal UV exposure are to be compared with each other, the need for intercomparability and intercalibration factors between different measurement systems becomes significant. (2) Three commonly used dosimeter types—(polysulphone film (PSF), biological, and electronic dosimeters)—were selected to perform intercalibration measurements. They differ in measurement principle and sensitivity, measurement accuracy, and susceptibility to inaccuracies. The aim was to derive intercalibration factors for these dosimeter types. (3) While a calibration factor between PSF and electronic dosimeters of about 1.3 could be derived for direct irradiation of the dosimeters, this was not the case for larger angles of incidence of solar radiation with increasing fractions of diffuse irradiation. Electronic dosimeters show small standard deviation across all measurements. For biological dosimeters, no intercalibration factor could be found with respect to PSF and electronic dosimeters. In a use case, the relation between steady-state measurements and personal measurements was studied. On average, persons acquired only a small fraction of the ambient radiation.

Background: Non-melanoma skin cancer (NMSC) represents the most frequently diagnosed cancer in humans. Occupational solar UV radiation exposure is associated with a higher-risk of developing NMSC, but still Romania does not acknowledge this affliction as an occupational disease. The study aims to determine if occupationally-induced NMSC is associated with more aggressive clinical and histopathological features compared to sporadic NMSC. Material and methods: A retrospective, analytical, comparative study was conducted during 2017-2019 in a University Department of Dermato-venereology in Bucharest, Romania, with focus on patients presenting with NMSC who underwent surgical excision of lesions followed by histopathological examination, classified as outdoor or indoor workers. High-risk clinical and histopathological characteristics were analysed and correlated with outdoor UV exposure. Outcomes: The study included 51 consecutive patients diagnosed with NMSC, of which 25 outdoor workers (OW) and 26 controls as indoor workers with no occupational UV exposure background. OW presented with 21 BCC and four SCC, while controls with 22 BCC and four SCC. Males were predominant in both groups and most patients came from urban environment. The mean age value was lower for the OW group compared to controls. OW had a 4.66 times higher risk of developing NMSC with aggressive location and size χ² (1, N=51) = 6.246, p=0.013, OR=4.66 (95% CI: 1.34, 16.23) and a 24-fold risk of developing NMSC with clinically poorly defined margins χ² (1, N=51) = 21.697, p

Occupational exposure to solar UV radiation (UVR) is relevant in the construction sector. We performed a one month monitoring period in late spring of direct individual solar UVR exposure measurements in a group of three construction workers from the Tuscany Italian region, using the GENESIS-UV system for personal dosimetry and for the collection of long-term exposure data in a centralized dataset. Then, we compared personal UVR exposure data with the environmental erythemal dose in the same days in clear-sky conditions. The individual solar UVR exposures of the construction workers in the period between 22nd May and 22nd June 2017 in Tuscany varied between 85.5 and 1556.8 J/m2, while the average daily exposure considering all the three workers resulted 573.3 J/m2. The percentage between individual vs environmental exposure varied from 2.7 up to 31.2%, resulting 12.2 on average. Of the total amount of 40 days/worker measured we detected exposure levels above the 100 J/m2 proposed limit value in 39 days/worker, with exceeding of the limit up to ten times per day, more than five times on average. In terms of cumulative UVR exposure, the construction workers received on average a total one month exposure, based on 21 working days, of 12 kJ/m2, and we estimated an annual exposure of about 70 kJ/m2, approximately corresponding to 700 Standard Erythemal Doses. Our data show that the exposure levels of the Italian construction workers are very high in late spring, systematically exceeding the limit values. These UVR exposure levels can be considered potentially dangerous, in particular if associated with relevant cumulative annual UV doses received at the body, which are related to an increased risk of adverse effects, including skin cancers. Accordingly, a big effort is needed, to urgently reduce occupational solar UVR exposure in the construction sector, improving the application of preventive measures.

INTRODUCTION: Understanding UV exposure is essential for the assessment of its contribution to the occupational risk of pilots developing ocular and skin pathologies. The objective of this observational study was to measure the UV exposure of pilots flying between the United Kingdom and a range of destinations at three different seasons.METHODS: The in-flight UV exposure of pilots was measured on 322 Monarch Airlines short-haul flights on the Airbus A321-231 and Airbus A320-214 to 31 destinations, mostly in Europe, from 4 UK airports in September 2016–August 2017. The erythema effective and UV-A doses were compared with the ICNIRP guidance and typical recreational weekend exposure of UK office workers.RESULTS: The erythema effective radiant doses did not exceed 0.1 SED. For most of the flights, the UV-A exposure was also low. On 27 single sector flights, UV-A exposure could have exceeded the ICNIRP guidance if eye protection was not used.DISCUSSION: The UV exposure in a cockpit is mostly governed by the presence of direct sunlight and the duration of a flight. The average monthly exposures were low and significantly below weekend recreational exposures of UK office workers over a similar period. To assess the contribution of occupational UV exposure to the risk of developing sun-related ocular and cutaneous pathologies, it is important to consider the accumulative flight time, destinations, and UV attenuation of aircraft windshields. Additionally, leisure and recreational outdoor time needs to be considered before meaningful overall risk analysis can be undertaken.Baczynska KA, Brown S, Chorley AC, O’Hagan JB, Khazova M, Lyachev A, Wittlich M. In-flight UV-A exposure of commercial airline pilots. Aerosp Med Hum Perform. 2020; 91(6):501–510.

A number of studies suggest that pilots are at twice the risk of melanoma and keratinocyte skin cancers than the general population, and that they have a raised mortality from melanoma. No conclusive links with in-flight exposure to ionising radiationor circadian rhythm disruption due to the pilots&rsquo, shift work were found. Possible over-exposure to ultraviolet radiation (UVR) may be implicated as pilots may be exposed to higher UV-A levels at cruise altitude compared with those at ground levels. The direct method of making in-flight spectral measurements has been carried out on a limited number of flights, but this technique is challenging, the use of small wearable sensors may be more appropriate but there are a few issues that should be addressed for their use in cockpit measurements. While the spectral response of sensors for erythema effective values usually closely matches the corresponding weighting function, the response of UV-A sensors may not be spectrally flat, which, if not corrected to account for the transmission of the aircraft windshield, could potentially result in large errors. In this paper, the spectral correction method was applied to the UV-A sensor of the Genesis-UV unit to measure UVR exposure of commercial pilots on 312 flights to a range of destinations from four UK airports from September 2016 to August 2017.

Background Squamous cell carcinoma (SCC) is among the most frequent types of cancer constituting a significant public health burden. Prevention strategies focus on limiting UV-exposure during leisure time. However, the relative impact of occupational and non-occupational UV-exposure for SCC occurrence is unclear. Objectives To investigate the association between occupational and non-occupational UV-exposure with SCC in a multicenter population-based case-control study hypothesizing that high occupational UV-exposure increases the risk for SCC. Methods Consecutive patients with incident SCC (n=632) were recruited from a German national dermatology network. Population-based controls (n=996) without history of skin cancer were recruited from corresponding residents’ registration offices and propensity score matched to cases. Lifetime UV-exposure, sociodemographic and clinical characteristics were assessed by trained physicians. Occupational and non-occupational UV-exposure dosages were estimated by blinded investigators using established reference values. Odds ratios (OR) and corresponding 95%-confidence intervals (95%-CI) were assessed using conditional logistic regression adjusting for relevant confounders. Results Total solar UV-exposure was significantly associated with an increased SCC. The OR (95%-CI) for high (>90th percentile) vs. low (

The aim of this study was to investigate the role of occupational and nonoccupational ultraviolet (UV)-exposure concerning the development of basal cell carcinoma (BCC).We undertook a population-based multicenter case-control study. Patients with first incident BCC (n = 836) were propensity score matched by age and sex to controls without skin cancer (n = 836). Sociodemographic characteristics, clinical characteristics, and lifetime UV-exposure were assessed by trained investigators. The differential estimation of occupational and nonoccupational UV-exposure dosages was based on validated instruments and established reference values. Associations were assessed using multivariable-adjusted conditional logistic regression models.Individuals with high levels of occupational UV-exposure were at significantly increased BCC-risk compared with individuals with low [odds ratio (OR) 1.84; 95% confidence interval (95% CI) 1.19 to 2.83 and moderate (OR 1.97; 95% CI 1.20 to 3.22) occupational UV-exposure. Nonoccupational UV-exposure was not independently associated with BCC.Skin cancer prevention strategies should be expanded to the occupational setting.

Objective This pilot study aimed to estimate annual occupational ultraviolet radiation (UVR) exposures based on 5 consecutive months of dosimetry measurements in Croatian construction workers and assess its relationship with the environmental data collected during the same period. Methods Four male construction industry workers from the vicinity of Zagreb (lat. 45.8° N, alt. 128 m) participated in the study. The study was a part of the GENESIS-UV project, attempting to collect long-term dosimetry data for outdoor occupations across Europe. Workers wore personal dosimeters (model X2012–10, Gigahertz, Tuerkenfeld, Germany) on the left upper arm during working hours in the period from June to October 2017. Median exposure assessment period was 57 working days. Results Estimates of annual occupational UVR exposure ranged from 76 SED to 976 SED per worker, reflecting large differences in patterns of workers' exposure. Daily UVR doses received by the workers were comparable to the values of other studies at similar latitudes, reaching up to 12 SED in summer months. Measurements of workers with high exposure values (estimated >600 SED/year) showed moderate to high associations with environmental UVR and insolation data (Spearman's correlation coefficient ranging from 0.57 to 0.73). Conclusions The results confirmed high occupational exposure of tested Croatian construction workers to solar UVR. However, more data are needed to explain the between-worker variability and develop prediction models with respect to specific work tasks. The use of environmental UVR for predicting long-term exposure should be considered, especially for high exposure tasks.

Background Work-related solar ultraviolet radiation (UVR) is an important factor in the pathogenesis of non-melanoma skin cancer (NMSC). The World Health Organization, through the International Agency for Research on Cancer, has classified solar UVR as a group 1 carcinogen since 2012. The main problems encountered so far in the study of occupationally induced skin cancer include the lack of accurate occupational UVR dosimetry as well as insufficient distinction between occupational and leisure UVR exposure and underreporting of NMSC. Objectives The aim of this study was to collect long-term individual UVR measurements in outdoor workers across European countries. Methods A prospective study was initiated through the European Academy of Dermatology and Venereology, Healthy Skin@Work Campaign, measuring UVR exposure doses at occupational settings of masons from five European countries. Measurements were performed for several consecutive months using the GENESIS-UV measurement system. Results The results identified alarming UVR exposure data. Average daily UVR doses ranged 148.40-680.48 J/m2 in Romania, 342.4-640.8 J/m2 in Italy, 165.5-466.2 J/m2 in Croatia, 41.8-473.8 J/m2 in Denmark and 88.15-400.22 J/m2 in Germany. Results showed an expected latitude dependence with increasing UVR yearly dosage from the north to the south of Europe. Conclusions This study shows that outdoor workers from EU countries included in this study are exposed to high levels of occupational solar UVR, vastly exceeding the occupational exposure limits for solar UVR exposure, considered to be 1-1.33 SED/day in the period from May to September. This finding may serve as an evidence-based recommendation to authorities on implementing occupational skin cancer prevention strategies.

Occupational exposure to solar UV radiation (SUVR), a Group 1 carcinogen according to the IARC classification is at high exposure levels in outdoor construction workers, usually above the suggested occupational limits. Furthermore, there are no regulations related to this exposure in the EU, except for the artificial UVR. Also, the use of the ICNIRP exposure guideline in an outdoor setting poses problems of adequate dose assessment. In this context, the main purpose of the study was to perform direct measurements of the SUVR dose in outdoor workers from the construction sector, using individual SUVR dosimeters (GENESIS-UV system), for a period of 7 months, from April to October, in a prospective, observational study in two groups of 10 outdoor workers in Romania, located at two different geographic sites. In term of cumulative standard erythema dose (SED), our study population of outdoor construction workers received high levels of solar UV radiation, ranging from 165 SED to 453 SED during 7 months of occupational activity, from April to October. Our results, ranging from 1.28 SED (standard erythema dose) per day to 6.4 SED per day pose an alarm signal to the national and European health authorities to take preventive action for outdoor workers, as the ICNIRP suggested limit value of 1.33 SED for mean daily erythemal UV exposure is vastly exceeded. We suggest that personal dosimetry for SUVR, from simple devices to complex systems as GENESIS-UV should be regularly and mandatory used in outdoor workers, similarly to the usage of personal dosimetry in occupational exposure to ionizing radiations, which could be included in European and national legislation to reduce both, the level of exposure and the detrimental effects on outdoor workers' health.

Background: The World Health Organization (WHO) and the International Labour Organization (ILO) are developing a joint methodology for estimating the national and global work-related burden of disease and injury (WHO/ILO joint methodology), with contributions from a large network of experts. Here, we present the protocol for two systematic reviews of parameters for estimating the number of disability-adjusted life years of cataracts from occupational exposure to solar ultraviolet radiation, to inform the development of the WHO/ILO joint methodology. Objectives: We aim to systematically review studies on occupational exposure to solar ultraviolet radiation (Systematic Review 1) and systematically review and meta-analyse estimates of the effect of occupational exposure to solar ultraviolet radiation on the development of cataract (Systematic Review 2), applying the Navigation Guide systematic review methodology as an organizing framework and conducting both systematic reviews in tandem and in a harmonized way. Data sources: Separately for Systematic Reviews 1 and 2, we will search electronic academic databases for potentially relevant records from published and unpublished studies, including Ovid Medline, PubMed, EMBASE, and Web of Sciences. We will also search electronic grey literature databases, Internet search engines and organizational websites; hand search reference list of previous systematic reviews and included study records; and consult additional experts. Study eligibility and criteria: We will include working-age (≥15 years) workers in WHO and/or ILO Member States, but exclude children (

Introduction We are exposed to solar ultraviolet radiation (UVR) every day, during work and leisure time. To date, there is only little knowledge about the level of irradiance. Already in 1992, UVR has been graded a group 1 carcinogen by the International Agency for Research on Cancer (IARC). In an ageing society, along with an ideal of a brown skin, non-melanoma skin cancer (NMSC) incidence rates rapidly increase. Methods For risk assessment and deduction of protective measures, real measured data serve best. Regarding solar UVR exposure, long-term personal dosimetry measurements is required. With GENESIS-UV, we designed a suitable system. With only little impairment, each test persons measures autonomously. Data are transferred via mobile service or internet to a data server once a week automatically. GENESIS-UV has proven to be feasible in collaborations in the whole world without any changes in the system. Results Since 2014, about 800 test persons delivered data points representing about 80 000 days of measurement – in Germany. We derived the annual irradiance for more than 100 occupations, and far more occupational activities as well. Interestingly, the irradiance values for the occupations cover a very wide range (www.dguv.de/genesis). Going deeper into the occupational activities, prevention-relevant information could be obtained. Since 2016, measurements have been expanded at European level. Discussion Outdoor workers have to be protected from UVR exposure. During working hours, their exposure can be up to 4.5 times the leisure dose. Our approach from occupations to occupational activities enables us to suggest very distinct protective measures, and drawing a full picture of workers exposure. Particularly, we showed that already short exposure times may lead to a notable hazard. Thus, the definition of an outdoor worker has to be reviewed. The study with GENESIS-UV is by far the largest study on UV irradiance of workers worldwide.

Introduction Electromagnetic fields (EMF) are everywhere. Most of our technology today and in future is based upon applications using electric currents, magnets and thus bear the risk to emit hazardous levels of electromagnetic fields. For this purpose workplace exposure due to EMF has to be assessed to ensure that impermissible exposure of employees is avoided. Particular attention has to be paid to employees with medical implants. Our presentation focusses on the process of exposure assessment of electromagnetic fields at several industrial workplaces. Methods Commonly measurements are performed to assess electromagnetic fields at workplace. Electromagnetic field simulations via numerical calculations can be needful if the EMF source is very complex or direct measurement is not possible. Measurements require a high degree of expert knowledge to assess the entire exposure scenario: relevant field sources have to be identified with both knowledge of fundamental functionality and operating frequency. Risk assessment usually considers working procedures and the whole working area at large. Results Several industrial applications imply a potential risk for safety and health of employees at their workplace. Thus, exposure has to be minimised by either technical, organisational and/or personal measures. Most of the required measures are simple as well as inexpensive, e.g. provision of operating instructions, safety instructions of employees, labelling of critical areas or just increase of distance to the field source. Only few cases require extensive measures, which may lead to a prohibition to resume work – especially for employees with active implants. Discussion EMF at workplace are underestimated and somehow neglected in the scope of risk assessment. That’s why people have to become sensitised to this topic to recognise possible hazards. Especially employees with medical implants are particularly vulnerable with regard to EMF. For this purpose a specially tailored risk assessment procedure is needed to address this problem completely and more adequate.

The HIV-1 encoded virus protein U (VpU) is required for efficient viral release from human host cells and for induction of CD4 degradation in the endoplasmic reticulum. The cytoplasmic domain of the membrane protein VpU (VpUcyt) is essential for the latter activity. The structure and dynamics of VpUcyt were characterized in the presence of membrane simulating dodecylphosphatidylcholine (DPC) micelles by high-resolution liquid state NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC micelles induces a well-defined membrane proximal α-helix (residues I39–E48) and an additional helical segment (residues L64–R70). A tight loop (L73–V78) is observed close to the C-terminus, whereas the interhelical linker (R49–E63) remains highly flexible. A 3D structure of VpUcyt in the presence of DPC micelles was calculated from a large set of proton–proton distance constraints. The topology of micelle-associated VpUcyt was derived from paramagnetic relaxation enhancement of protein nuclear spins after the introduction of paramagnetic probes into the interior of the micelle or the aqueous buffer. Qualitative analysis of secondary chemical shift and paramagnetic relaxation enhancement data in conjunction with dynamic information from heteronuclear NOEs and structural insight from homonuclear NOE-based distance constraints indicated that micelle-associated VpUcyt retains a substantial degree of structural flexibility.

The human immunodeficiency virus type 1 (HIV-1) protein U (VpU) is an accessory protein responsible for enhancement of viral particle release and down regulation of the T-lymphocyte coreceptor CD4. Direct binding between the cytoplasmic domains of CD4 and VpU as well as phosphorylation of serines 53 and 57 in the cytoplasmic domain of VpU plays a central role in CD4 downregulation. We investigated structural consequences of phosphorylation of the two serines using nuclear magnetic resonance spectroscopy. A uniformly 15N and 13C stable isotope-labeled 45-residue peptide comprising the cytoplasmic domain of VpU (VpUcyt) was recombinantly produced in E .coli. The peptide forms two helices (commonly referred to as helix 2 and 3) in the presence of membrane mimicking dodecylphosphocholine (DPC) micelles, which flank a flexible region containing the two phosphorylation sites. Phosphorylation does not cause any drastic structural changes in the secondary structure of VpUcyt. However, an N-terminal elongation of helix 3 and a slightly reduced helicity at the C-terminus of helix 2 are observed upon phosphorylation based on characteristic changes of 13Cα and 13Cβ chemical shifts. Phosphorylation also reduces the local mobility of the protein backbone in the loop region containing the phosphorylation sites according to heteronuclear 1H15N nuclear Overhauser enhancement (NOE) data. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton–proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.

HIV-1 Vpu and CD4(372–433), a peptide comprising the transmembrane and cytoplasmic domain of human CD4, were recombinantly expressed in Escherichia coli, uniformly labeled with 13C and 15N isotopes, and separately reconstituted into phospholipid bilayers. Highly resolved dipolar cross-polarization (CP)-based solid-state NMR spectra of the two transmembrane proteins were recorded under magic angle sample spinning. Partial assignment of 13C resonances was achieved. Site-specific assignments were obtained for 13 amino acid residues of CD4(372–433) and two Vpu residues. Additional amino acid type-specific assignments were achieved for 10 amino acid spin systems for both CD4(372–433) and Vpu. Further, structural flexibility was probed with different dipolar recoupling techniques, and the correct insertion of the transmembrane domains into the lipid bilayers was confirmed by proton spin diffusion experiments.

Viral protein U (VpU) of HIV-1 plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. In this study, regions in the cytoplasmic domain of VpU involved in CD4 binding were identified by NMR spectroscopy. Amino acids in both helices found in the cytoplasmic region of VpU in membrane-mimicking detergent micelles experience chemical shift perturbations upon binding to CD4, whereas amino acids between the two helices and at the C-terminus of VpU show no or only small changes, respectively. The topology of the complex was further studied with paramagnetic relaxation enhancement. Paramagnetic spin labels were attached at three sequence positions of a CD4 peptide comprising the transmembrane and cytosolic domains of the receptor. VpU binds to a membrane-proximal region in the cytoplasmic domain of CD4. Structured digital abstract VpU and CD4 bind by nuclear magnetic resonance (View interaction)

Human CD4 is a 433 residue transmembrane protein involved in the body's adaptive immune response. The extracellular domain of CD4 serves as primary receptor of the human immunodeficiency virus (HIV-1) and binds the viral glycoprotein gp120. The cytoplasmic domain of the 81 residue Virus protein U (VpU) plays an important role in downregulation of CD4. It directly binds the cytoplasmic domain of CD4 in the endoplasmic reticulum, which initiates a series of events resulting in CD4 degradation in the proteasome. We characterized the CD4 - VpU interaction by liquid state NMR spectroscopy utilizing paramagnetic relaxation enhancement (PRE). The PRE effect is a distance-dependent enhancement of the spin relaxation in magnetically active nuclei in the vicinity of a paramagnetic centre. PRE leads to line broadening or complete quenching of NMR signals and indicates spatial proximity to the probe. We recorded NMR spectra of the 15N-labelled cytoplasmic domain of VpU in the presence of varying amounts of a single-cysteine-mutant of CD4(372-433), which was labelled with the active PRE-probe, methanethiosulfonate (MTSL). This CD4 polypeptide contains the single transmembrane and the C-terminal cytoplasmic domain of CD4. Experiments were conducted in membrane-mimicking dodecyl phosphocholine (DPC) micelles. Spectra recorded in the presence of paramagnetic MTSL show reduced NMR signal intensities of certain amino acids of 15N-labeled VpU. Additionally, we studied chemical shift perturbations (CSP's) of VpU resonances observed on titrating increasing concentrations of unlabelled CD4. Such chemical shift changes are expected for residues in the binding interface but may also occur at remote sites due to allosteric effects. Our data reveal VpU residues involved in CD4 binding, provide insight into the exchange regime, and yield an estimate of the binding affinity.

The HIV-1 encoded virus protein U (VpU) is required for efficient viral release from human host cells and for induction of CD4 degradation in the endoplasmic reticulum. The cytoplasmic domain of the membrane protein VpU (VpUcyt) is essential for the latter activity. The structure and dynamics of VpUcyt were characterized in the presence of membrane simulating dodecylphosphatidylcholine (DPC) micelles by high-resolution liquid state NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC micelles induces a well-defined membrane proximal alpha-helix (residues I39-E48) and an additional helical segment (residues L64-R70). A tight loop (L73-V78) is observed close to the C-terminus, whereas the interhelical linker (R49-E63) remains highly flexible. A 3D structure of VpUcyt in the presence of DPC micelles was calculated from a large set of proton-proton distance constraints. The topology of micelle-associated VpUcyt was derived from paramagnetic relaxation enhancement of protein nuclear spins after the introduction of paramagnetic probes into the interior of the micelle or the aqueous buffer. Qualitative analysis of secondary chemical shift and paramagnetic relaxation enhancement data in conjunction with dynamic information from heteronuclear NOEs and structural insight from homonuclear NOE-based distance constraints indicated that micelle-associated VpUcyt retains a substantial degree of structural flexibility.

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4+ cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes. Besides its cellular functions, CD4 serves as the main receptor for human immunodeficiency virus type I (HIV-1). During T-cell infection, the CD4 extracellular domain is bound by HIV-1 gp120, the viral surface glycoprotein, which triggers a number of conformational changes ultimately resulting in virion entry of the cell. Subsequently, CD4 is downregulated in infected cells by multiple strategies that involve direct interactions of the HIV-1 proteins VpU and Nef with the cytoplasmic part of CD4. In the present work, we describe the NOE-based solution structure of the transmembrane and cytoplasmic domains of the cystein-free variant of CD4 (CD4mut) in dodecylphosphocholine (DPC) micelles. Furthermore, we have characterized micelle-inserted CD4mut by paramagentic relaxation enhancement (PRE) agents and 1H-15N heteronuclear NOE data. CD4mut features a stable and well-defined transmembrane helix from M372 to V395 buried in the micellar core and a cytoplasmic helix ranging from A404 to L413. Experimental data suggest the amphipathic cytoplasmic helix to be in close contact with the micellar surface. The role of the amphipathic helix and its interaction with the micellar surface is discussed with respect to the biological function of the full-length CD4 protein.

The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Here we report heterologous expression and purification of a CD4 fragment comprising the transmembrane and cytoplasmic domains of human CD4. A synthetic gene encoding CD4 amino acid residues 372-433 and a protease cleavage site was cloned into the pTKK19xb/ub plasmid. The CD4 fragment was expressed in Escherichia coli C43(DE3) cells as a ubiquitin fusion with an N-terminal His-tag, isolated, released by PreScission proteolytic cleavage, and purified to homogeneity. Incorporation of the recombinant CD4 fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. A high resolution NMR spectrum of uniformly (15)N-labeled CD4 peptide in membrane simulating micelles proves the possibility of solution NMR studies of this CD4 fragment and of its molecular complexes.

Insolubility of integral membrane proteins (IMPs) in buffer often prevents their investigation with biophysical methods developed for soluble proteins. First, solution NMR of large proteins or complexes is limited by slow rotational diffusion. IMPs in cellular membranes or liposomes are beyond the size limit of solution NMR. Therefore, detergent micelles or bicelles are often used for solubilization and NMR studies of IMPs. Unfortunately, detergents may destabilize the native structure or compromise the activity of proteins. Nanodiscs are a lipid-based, detergent free, relatively small and very promising new model membrane system. We inserted a recombinantly produced and 15N, 13C-labeled fragment of human CD4 into nanodiscs. This polypeptide comprises the membrane-spanning and the cytoplasmic domains of CD4. Our NMR data demonstrate the feasibility of solution NMR spectroscopy on IMPs in nanodiscs. Second, surface plasmon resonance (SPR) is predominantly utilized in interaction studies of soluble proteins. Incorporation of IMPs into nanodiscs provides a close to native environment to the membrane protein and results in a water-soluble proteolipid particle that might be amenable to standard SPR-based methodology. We reconstituted a decahistidine-tagged IMP into nanodiscs and studied binding between the nanodisc-inserted IMP and a PentaHis monoclonal antibody (mAb) immobilized on the surface of a CM5-sensorchip. For comparison, we also determined the affinity of the decahistidine-tagged soluble domain of the same IMP toward the immobilized PentaHis mAb. Binding affinities were almost identical in both cases. However, the association and dissociation rate constants were found to differ, which is in agreement with the distinct diffusion coefficients of the soluble analyte particles. Our data indicate that nanodisc-inserted IMPs can serve as analyte in interaction studies of membrane proteins.