Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis

Protein phosphorylation is the most abundant post-translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage-displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild-type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr-binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome. Statement: Src Homology 2 (SH2) domains are modular domains that recognize phosphorylated tyrosine embedded in proteins, transducing these post-translational modifications into cellular responses. Here we used phage display to engineer hundreds of SH2 domain variants with altered binding specificities and enhanced affinities, which enabled efficient and differential enrichment of the human phosphoproteome for analysis by mass spectrometry. These engineered SH2 domain variants will be useful tools for elucidating the molecular determinants governing SH2 domains binding specificity and for enhancing analysis and understanding of the human phosphoproteome

How can we treat vulvar carcinoma in pregnancy? A systematic review of the literature

According to our systematic literature review (PRISMA guidelines), only 37 vulvar squamous cell carcinomas (VSCCs) were diagnosed during pregnancy (age range: 17\u201341 years). The tumor size range was 0.3\u201315 cm. The treatment was performed after (14/37, 38%), before (10/37, 27%), or before\u2010and\u2010after delivery (11/37, 30%). We found that 21/37 (57%) cases were stage I, 2 II (5%), 11 III (30%), and 3 IVB (8%). HPV\u2010related features (condylomas/warts; HPV infection; high\u2010grade squamous intraepithelial lesion) were reported in 11/37 (30%) cases. We also found that 9/37 (24%) patients had inflammatory conditions (lichen sclerosus/planus, psoriasis, chronic dermatitis). The time\u2010to\u2010recurrence/progression (12/37, 32%) ranged from 0 to 36 (mean 9) months. Eight women died of disease (22%) 2.5\u201348 months after diagnosis, 2 (5%) were alive with disease, and 23 (62%) were disease\u2010free at the end of follow\u2010up. Pregnant patients must be followed\u2010up. Even if they are small, newly arising vulvar lesions should be biopsied, especially in women with risk factors (HPV, dermatosis, etc.). The treatment of VSCCs diagnosed in late third trimester might be delayed until postpartum. Elective cesarean section may prevent vulvar wound dehiscence. In the few reported cases, pregnancy/fetal outcomes seemed to not be affected by invasive treatments during pregnancy. However, clinicians must be careful; larger cohorts should define the best treatment. Definite guidelines are lacking, so a multidisciplinary approach and discussion with patients are mandatory

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing

Modular assembly of proteins on nanoparticles

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond

Kidney and urinary tract polyomavirus infection and distribution: molecular biology investigation of 10 consecutive autopsies

CONTEXT: Distinct human polyomavirus genotypes cause different diseases in patients with renal transplants: BK virus (BKV) causes tubulointerstitial nephritis and ureteral stenosis, whereas both JC virus (JCV) and BKV are responsible for hemorrhagic cystitis. These findings could result from a selective infection of kidney and urinary tract segments by JCV or BKV. OBJECTIVE: To verify this hypothesis, 10 complete, unselected, consecutive autopsies from 9 immunocompetent patients and 1 patient affected by acquired immunodeficiency syndrome were investigated. DESIGN: Samples from kidneys (n = 80), renal pelvis (n = 20), ureter (n = 40), and urinary bladder (n = 30) obtained from 10 consecutive autopsies were investigated by means of multiplex nested polymerase chain reaction to detect polyomavirus DNA and to distinguish different species of the Polyomavirus genus. In situ hybridization and immunohistochemistry were also carried out to define the viral status of the infected tissues. RESULTS: Polyomavirus DNA was detected in all of the subjects (positive samples ranging from 2 to 7 samples), for a total of 43 of 170 samples (25.3%), distributed as follows: urinary bladder (10/30, 33%), renal pelvis (6/20, 30%), ureter (10/40, 25%), and kidney tissue (17/80, 21%). We found that JCV was most frequently detected overall (23/43 samples, 53.5%) and was also detected most frequently within the kidney (8/17 positive samples, 47%), the renal pelvis (5/6 positive samples, 70%), and the ureter (7/10 positive samples, 70%), whereas BKV was found in 14 samples (32.5%), and it was the prevailing genotype in urinary bladder (6/10 positive samples, 60%). Coinfection of BKV-JCV was found in 6 samples (14%). Immunohistochemistry and in situ hybridization returned negative results. CONCLUSIONS: The viruses JCV and BKV latently persist randomly in kidney and urinary tract. Distinct diseases induced by them could be related more closely to molecular viral rearrangements than to the topographic distribution of latent viruses

Are sequence variations in the BK virus control region essential for the development of polyomavirus nephropathy?

BK virus replication is regulated by the noncoding control region (NCCR); major NCCR rearrangements could modify the strength of viral replication, having a role in the development of polyomavirus-associated nephropathy (PAN). Urine (n = 34), blood (n = 32), and renal biopsy samples (n = 13) from 5 transplant recipients with PAN underwent nested polymerase chain reaction to search for the NCCR region. Sequence analysis was performed on all NCCR fragments obtained. Decoy cells were evaluated semiquantitatively in urine and PAN staged in renal biopsy specimens; the results were related to the presence and type of NCCR sequence variations. Major NCCR rearrangements were found in urine (9/75 [12%]), blood (7/30 [23%]), and renal biopsy (4/15 [27%]) samples in 3 cases; 2 cases had only unrearranged strains. Neither the detection and number of decoy cells nor the PAN stage were related to the specific type of NCCR sequence rearrangements. NCCR rearrangements do not seem essential for the development of PAN