To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of noncovalent interactions that allosterically couple the two SH2 domains during binding to doubly phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.

The T‐Cell signaling commences with the binding of Antigen Presenting Cell with T‐cell Receptor (TCR) Complex leading to the recruitment of a non‐receptor tyrosine kinase, Zeta Associated Protein‐70 (ZAP‐70), to the membrane. ZAP‐70 is comprised of the tandem SH2 domain (tSH2), which is made up of N‐terminal SH2 (N‐SH2) and C‐terminal SH2 (C‐SH2) domains, and a carboxy‐terminal kinase domain. The two SH2 domains cooperatively bind to the doubly‐phosphorylated ITAM (ITAM‐Y2P) motif resulting in a conformational rearrangement in the whole of the regulatory module, thereby releasing the autoinhibitory conformation of the kinase domain. However, the structural mechanism of how the two SH2 domain cross‐talk during ITAM‐Y2P binding and dissociation from the membrane are poorly understood. We observed that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly‐phosphorylated ITAM‐ ζ1 (ITAM‐Y2P‐ ζ1) peptide. The C‐SH2 binds first to one of the phosphotyrosine in ITAM‐Y2P motifs, leading to the formation of an encounter complex which further leads to the binding by N‐SH2 domain. NMR titration and Network analysis studies show that an intra‐residue network, which allosterically interconnects the N‐SH2 and C‐SH2 binding sites, plays a central role in ITAM‐Y2P binding. Further, a mutation in the allosteric network (F117A, W165C) uncouples the two binding sites, causing autoimmune arthritis in SKG mice. The allosteric coupling in ZAP‐70 is found to be unique and thus plays a significant role in T‐cell regulation. Support or Funding Information Authors are thankful to Prof. John Kuriyan and Prof. David E. Wemmer for access to the 900 MHz NMR spectrometer at the University of California, Berkeley. Dr. Jeffrey G. Pelton and Dr. Patrick R. Visperas at the University of California, Berkeley for NMR data collection and sample preparation. Authors thank Prof. Gautam Basu and Mr. Barun Majumder at Bose Institute, India for access to 700 MHz NMR spectrometer. Authors are thankful to Dr. Ashima Bhattacharjee, Dr. Pradip K. Tarafdar, and Prof. Pradipta Purkayastha for access to ITC and fluorimeter. Authors thank Prof. Giuseppe Melacini and Prof. Maitrayee DasGupta for helpful discussion. The authors thanks research funding from IISER Kolkata, infrastructural facilities supported by IISER Kolkata and DST‐FIST (SR/FST/LS‐II/2017/93(c)). This work is supported by grant from SERB (ECR/2015/000142) and DBT Ramalingaswami Fellowship (BT/RFF/Re‐entry/14/2014) to Rahul Das.

The level and type of fungal infection on grains varying in hardness at different stages of development over two seasons has been studied. Hard grains showed less incidence of grain moulds than soft grains during development. Microscopic examination showed more intense deposition of protein bodies in hard than in the soft grains. The presence of fungal hyphae in the endosperm of soft grains and pitted starch granules was clearly visible microscopically. Extracts of immature and mature hard and soft endosperm were inhibitory to Fusarium monififorme growth. These inhibitors were heat labile and non-dialysable indicating that protein factors may be involved. The activities of inhibitors to serine proteases were comparatively higher in endosperm of hard grains during development. The endosperm of hard grains contained more protein and prolamine than that of soft grains. A role for proteins in resistance of fungal infection in low tannin lines is presented.

No abstract

Three proteins potently inhibitory to the growth of Fusarium moniliforme, the grain mould pathogen have been identified from sorghum endosperm, using aqueous extraction, gel filtration and ion-exchange chromatography coupled with bioassay. The aqueous extract (Tris buffer, pH 6.8) of sorghum endosperm flour was separated into two fractions by passing through Sephadex G-50. The void volume fraction, showing antifungal activity was further fractionated by ion-exchange chromatography on SP Sephadex C-50. The most potent fraction was that which bound to the SP Sephadex column at pH 6.0. A less potent fraction with an 18 kDa protein as major component was released on shifting the pH to 8.5. The bound fraction at pH 8.5 was a much more potent inhibitor of fungal growth than the unbound fraction; and it was composed of two major polypeptides with molecular weights of 26 and 30 kDa. All three polypeptides were separated on SDS-PAGE gels, transferred onto Immobilon and tested for antifungal activity. The test showed that the 26 and 30 kDa proteins retained the antifungal activity even after electrotransfer. Antibodies raised against the electroeluted proteins were shown to abolish the antifungal activity of the endosperm extracts. These proteins appeared only in the endosperm of sorghum grains as revealed by dot immunobinding assay. The 26 and 30 kDa antifungal proteins were also present in pearl millet and maize, while the 18 kDa protein was found only in sorghum. Cross-reactivity of antibodies with the aqueous extracts of wheat, rice and ragi was not seen.

Apoptosis was induced in HeLa cells by exposure to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for various time intervals (up to 120 min). Apoptotic death was confirmed by the microscopic observation of cell blebbing, cell granulation, and cell aggregation. Cells also showed loss of phospholipid symmetry as judged by immunofluorescent microscopy with fluorescently labeled phosphatidyl serine-specific annexin V. In addition, staining of cells with ethidium bromide showed the presence of genomic DNA apoptotic bodies. The protein expression levels of c-jun and c-fos increased in DNA-damaged HeLa cells after MNNG treatment in a time-dependent fashion. Although the levels of c-fos increased rapidly during the first 30 min and remained high for 2 hr, the increase in c-jun expression was more gradual and slower (60-120 min) after MNNG treatment. These results are consistent with the conclusion that c-fos is important in the initial stages (commitment phase) of apoptosis and c-jun is involved in the late stages (execution phase) of apoptosis induced with alkylating agents.

22T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a 23 non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to 24 the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 25 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results 26show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-27 phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine 28 residue of ITAM peptide to form an encounter complex leading to subsequent binding of second 29 phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that 30 allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in 31 the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to 32 the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma 33 membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is 34 unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in 35 B-cells. 36Significance 37 38 T-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and 39 Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet 40 the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand, 41whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-42 70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is 43 unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while 44 binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism 45 of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant 46 found in SKG mice. 47 48 49 50 51The zeta-chain-associated protein tyrosine kinase, ZAP-70, is a non-receptor tyrosine kinase 52 crucial for T-cell signaling, development, activation, and proliferation(1-4). T-cell signaling is 53 commenced by the recruitment of two protein tyrosine kinase, Src family kinase Lck and ZAP-70, to the 54 activated molecular complex of T-cell antigen receptor (TCR)(5, 6). Lck, phosphorylate several tyrosine 55 residues of the immuno-receptor tyrosine-based activation motifs (ITAM) on the intracellular segment 56 of CD3 heterodimer (made up of d, g, and e) and ζ homodimer associated with the TCR(5, 7-10). ZAP-57 70 is spontaneously recruited to the membrane by binding to the doubly-phosphorylated ITAM (ITAM-58 Y2P) motifs (11)(12)(13)(14). Recruitment of ZAP-70 allows phosphorylation of scaffold proteins that initiates a 59 cascade of downstream biological events (15, 16)...